U.S. patent application number 10/603891 was filed with the patent office on 2004-12-16 for markers for differential diagnosis and methods of use thereof.
This patent application is currently assigned to Biosite Incorporated. Invention is credited to Anderberg, Joseph Michael, Buechler, Kenneth F., Dahlen, Jeffrey R., Kirchick, Howard J., Maisel, Alan, McPherson, Paul H..
Application Number | 20040253637 10/603891 |
Document ID | / |
Family ID | 33515036 |
Filed Date | 2004-12-16 |
United States Patent
Application |
20040253637 |
Kind Code |
A1 |
Buechler, Kenneth F. ; et
al. |
December 16, 2004 |
Markers for differential diagnosis and methods of use thereof
Abstract
The present invention provides methods for the identification
and use of diagnostic markers for differential diagnosis of
diseases. In various aspects, the invention relates to methods and
compositions able to determine the presence or absence of one, and
preferably a plurality, of diseases that exhibit one or more
similar or identical symptoms. Such methods and compositions can be
used to provide assays and assay devices for use in determining the
disease underlying one or more non-specific symptoms exhibited in a
clinical setting.
Inventors: |
Buechler, Kenneth F.;
(Rancho Santa Fe, CA) ; Maisel, Alan; (Del Mar,
CA) ; Anderberg, Joseph Michael; (Encinitas, CA)
; McPherson, Paul H.; (Encinitas, CA) ; Dahlen,
Jeffrey R.; (San Diego, CA) ; Kirchick, Howard
J.; (San Diego, CA) |
Correspondence
Address: |
FOLEY & LARDNER
P.O. BOX 80278
SAN DIEGO
CA
92138-0278
US
|
Assignee: |
Biosite Incorporated
|
Family ID: |
33515036 |
Appl. No.: |
10/603891 |
Filed: |
June 24, 2003 |
Related U.S. Patent Documents
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Application
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10603891 |
Jun 24, 2003 |
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10410572 |
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10330696 |
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PCT/US02/26604 |
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10603891 |
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PCT/US02/14219 |
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10139086 |
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PCT/US02/11411 |
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09835298 |
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60313775 |
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60334964 |
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60313775 |
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60315642 |
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Current U.S.
Class: |
435/7.1 ;
436/518 |
Current CPC
Class: |
A61B 5/14546 20130101;
A61B 5/412 20130101; G01N 2800/324 20130101; G01N 2800/2871
20130101; A61B 5/4076 20130101; G01N 33/6896 20130101; G01N 2800/60
20130101 |
Class at
Publication: |
435/007.1 ;
436/518 |
International
Class: |
G01N 033/53 |
Claims
We claim:
1. A method of analyzing a subject sample for a plurality of
subject-derived markers selected to distinguish amongst a plurality
of cardiovascular disorders, comprising: assaying said sample for
the presence or amount of one or more subject-derived markers
related to blood pressure regulation, and for the presence or
amount of one or more subject-derived markers related to myocardial
injury, and characterizing said subject's risk of having developed
or of developing each of said plurality of cardiovascular disorders
based upon the presence or amount of said markers, wherein the
amount of at least one of said one or more subject-derived markers
is not compared to a predetermined threshold amount.
2. A method according to claim 1, wherein said characterization
step is performed without comparing the amount of any of said
markers to a predetermined threshold amount.
3. A method according to claim 1, wherein said subject-derived
marker(s) related to blood pressure regulation are selected from
the group consisting of B-type natriuretic peptide, a marker
related to B-type natriuretic peptide, C-type natriuretic factor,
urotensin II, arginine vasopressin, aldosterone, angiotensin I,
angiotensin II, angiotensin III, bradykinin, calcitonin,
procalcitonin, calcitonin gene related peptide, adrenomedullin,
calcyphosine, endothelin-2, endothelin-3, rennin, A-type
natriuretic peptide, and urodilatin, and wherein said
subject-derived marker(s) related to myocardial injury are selected
from the group consisting of free cardiac troponin I, free cardiac
troponin T, cardiac troponin I in a complex comprising one or both
of troponin T and troponin C, cardiac troponin T in a complex
comprising one or both of troponin I and troponin C, free and
complexed cardiac troponin I, free and complexed cardiac troponin
T, creatine kinase-MB, myoglobin, glycogen phosphorylase-BB,
annexin B, P-enolase, heart-type fatty acid binding protein, and
S-100ao.
4. A method according to claim 3, wherein said method comprises
assaying said sample for the presence or amount of B-type
natriuretic peptide or a marker related to B-type natriuretic
peptide, creatine kinase-MB, total cardiac troponin I, and
myoglobin.
5. A method according to claim 1, wherein said method further
comprises assaying said sample for the presence or amount of one or
more subject-derived markers related to inflammation.
6. A method according to claim 5, wherein said characterization
step is performed without comparing the amount of any of said
marker(s) related to inflammation to a predetermined threshold
amount.
7. A method according to claim 5, wherein said marker(s) related to
inflammation are selected from the group consisting of C-reactive
protein, an interleukin, interleukin-1 receptor agonist, CD54,
CD106, monocyte chemotactic protein-1, caspase-3, lipocalin-type
prostaglandin D synthase, mast cell tryptase, eosinophil cationic
protein, KL-6, haptoglobin, tumor necrosis factor .alpha., tumor
necrosis factor .beta., fibronectin, and vascular endothelial
growth factor.
8. A method according to claim 7, wherein said method comprises
assaying said sample for the presence or amount of B-type
natriuretic peptide or a marker related to B-type natriuretic
peptide, creatine kinase-MB, total cardiac troponin I, myoglobin,
and C-reactive protein.
9. A method according to claim 1, wherein said method further
comprises assaying said sample for the presence or amount of one or
more subject-derived markers related to coagulation and
hemostasis.
10. A method according to claim 9, wherein said characterization
step is performed without comparing the amount of any of said
marker(s) related to coagulation and hemostasis to a predetermined
threshold amount.
11. A method according to claim 9, wherein said subject-derived
marker(s) related to coagulation and hemostasis are selected from
the group consisting of plasmin, fibrinogen, D-dimer,
.beta.-thromboglobulin, platelet factor 4, fibrinopeptide A,
platelet-derived growth factor, prothrombin fragment 1+2,
plasmin-.alpha.2-antiplasmin complex, thrombin-antithrombin III
complex, P-selectin, thrombin, von Willebrand factor, tissue
factor, and thrombus precursor protein.
12. A method according to claim 11, wherein said method comprises
assaying said sample for the presence or amount of B-type
natriuretic peptide or a marker related to B-type natriuretic
peptide, D-dimer, creatine kinase-MB, total cardiac troponin I, and
myoglobin.
13. A method according to claim 5, wherein said method further
comprises assaying said sample for the presence or amount of a
subject-derived marker related to coagulation and hemostasis.
14. A method according to claim 13, wherein said method comprises
assaying said sample for the presence or amount of B-type
natriuretic peptide or a marker related to B-type natriuretic
peptide, D-dimer, creatine kinase-MB, total cardiac troponin I,
myoglobin, and C-reactive protein.
15. A method according to claim 1, wherein said subject sample is
selected from the group consisting of a blood sample, a serum
sample, and a plasma sample.
16. A method according to claim 1, wherein said plurality of
cardiovascular disorders are selected from the group consisting of
myocardial infarction, congestive heart failure, acute coronary
syndrome, unstable angina, and pulmonary embolism.
17. A method according to claim 1, wherein said correlating step
comprises comparing at least one marker amount to a predetermined
threshold level.
18. A test device for performing the method of claim 1, comprising:
a test surface comprising a plurality of discrete addressable
locations corresponding to said plurality of subject-derived
markers, each said location comprising an antibody immobilized at
said location selected to bind for detection one of said plurality
of subject-derived markers.
19. A method of analyzing a subject sample for a plurality of
subject-derived markers selected to distinguish amongst a plurality
of cerebrovascular disorders, comprising: assaying said sample for
the presence or amount of one or more subject-derived markers
related to blood pressure regulation, and for the presence or
amount of one or more subject-derived markers related to neural
tissue injury, and characterizing said subject's risk of having
developed or of developing each of said plurality of
cerebrovascular disorders based upon the presence or amount of said
markers, wherein the amount of one or more of said markers is not
compared to a predetermined threshold amount.
20. A method according to claim 19, wherein said characterization
step is performed without comparing the amount of any of said
markers to a predetermined threshold amount.
21. A method according to claim 19, wherein said subject-derived
marker(s) related to blood pressure regulation are selected from
the group consisting of B-type natriuretic peptide, a marker
related to B-type natriuretic peptide, C-type natriuretic factor,
urotensin II, arginine vasopressin, aldosterone, angiotensin I,
angiotensin II, angiotensin III, bradykinin, calcitonin,
procalcitonin, calcitonin gene related peptide, adrenomedullin,
calcyphosine, endothelin-2, endothelin-3, rennin, A-type
natriuretic peptide, and urodilatin, and wherein said
subject-derived marker(s) related to neural tissue injury are
selected from the group consisting of precerebellin 1, cerebillin
1, cerebellin 3, chimerin 1, chimerin 2, calbrain, calbindin D,
brain tubulin, brain fatty acid binding protein ("B-FABP"), brain
derived neurotrophic factor ("BDNF"), carbonic anhydrase XI,
CACNA1A calcium channel gene, nerve growth factor .beta., atrophin
1, apolipoprotein E4-1, protein 4.1B, 14-3-3 protein, ciliary
neurotrophic factor, creatine kinase-BB, C-tau, glial fibrillary
acidic protein ("GFAP"), neural cell adhesion molecule ("NCAM"),
neuron specific enolase, S-100b, prostaglandin D synthase,
neurokinin A, neurotensin, and secretagogin.
22. A method according to claim 19, wherein said method further
comprises assaying said sample for the presence or amount of one or
more subject-derived markers related to inflammation.
23. A method according to claim 22, wherein said characterization
step is performed without comparing the amount of any of said
marker(s) related to inflammation to a predetermined threshold
amount.
24. A method according to claim 19, wherein said method further
comprises assaying said sample for the presence or amount of one or
more subject-derived markers related to coagulation and
hemostasis.
25. A method according to claim 24, wherein said characterization
step is performed without comparing the amount of any of said
marker(s) related to coagulation and hemostasis to a predetermined
threshold amount.
26. A method according to claim 19, wherein said method further
comprises assaying said sample for the presence or amount of one or
more subject-derived markers related to apoptosis.
27. A method according to claim 26, wherein said subject-derived
marker(s) related to apoptosis are selected from the group
consisting of spectrin, cathepsin D, caspase 3, s-acetyl
glutathione, and ubiquitin fusion degradation protein 1
homolog.
28. A method according to claim 27, wherein said characterization
step is performed without comparing the amount of any of said
marker(s) related to apoptosis to a predetermined threshold
amount.
29. A method according to claim 19, wherein said method further
comprises assaying said sample for the presence or amount of one or
more subject-derived acute phase markers.
30. A method according to claim 29, wherein said subject-derived
acute phase marker(s) are selected from the group consisting of
hepcidin, HSP-60, HSP-65, HSP-70, S-FAS ligand, asymmetric
dimethylarginine, matrix metalloproteins 11, 3, and 9, defensin HBD
1, defensin HBD 2, serum amyloid A, oxidized LDL, insulin like
growth factor, transforming growth factor .beta., e-selectin,
glutathione-S-transferase, hypoxia-inducible factor-1.alpha.,
inducible nitric oxide synthase, intracellular adhesion molecule,
lactate dehydrogenase, monocyte chemoattractant peptide-1, n-acetyl
aspartate, prostaglandin E2, receptor activator of nuclear factor
ligand, TNF receptor superfamily member 1A, TNF.alpha., vascular
cell adhesion molecule, and cystatin C.
31. A method according to claim 27, wherein said characterization
step is performed without comparing the amount of any of said acute
phase marker(s) to a predetermined threshold amount.
32. A method according to claim 19, wherein said method comprises
assaying said sample for the presence or amount of B-type
natriuretic peptide or a marker related to B-type natriuretic
peptide, caspase-3, interleukin-8, creatine kinase-BB, C-reactive
protein, S-100b, matrix metalloprotein-9, and neural cell adhesion
molecule.
33. A method according to claim 19, wherein said plurality of
cerebrovascular disorders are selected from the group consisting of
ischemic stroke, hemorrhagic stroke, transient ischemic attack, and
subarachnoid hemorrhage.
34. A method according to claim 19, wherein said subject sample is
selected from the group consisting of a blood sample, a serum
sample, and a plasma sample.
35. A test device for performing the method of claim 19,
comprising: a test surface comprising a plurality of discrete
addressable locations corresponding to said plurality of
subject-derived markers, each said location comprising an antibody
immobilized at said location selected to bind for detection one of
said plurality of subject-derived markers.
36. A method of analyzing a subject sample for a plurality of
subject-derived markers selected to identify subjects suffering
from myocardial infarction, comprising: assaying said sample for
the presence or amount of a plurality of subject-derived marker(s)
related to myocardial injury selected from the group consisting of
free cardiac troponin I, free cardiac troponin T, cardiac troponin
I in a complex comprising one or both of troponin T and troponin C,
cardiac troponin T in a complex comprising one or both of troponin
I and troponin C, free and complexed cardiac troponin I, free and
complexed cardiac troponin T, creatine kinase-MB, myoglobin,
glycogen phosphorylase-BB, annexin B, .beta.-enolase, heart-type
fatty acid binding protein, and S-100ao, and characterizing said
subject's risk of having suffered a myocardial infarction based
upon the presence or amount of said markers, wherein the amount of
each of said markers is not compared to a predetermined threshold
amount.
Description
[0001] This application is a continuation in part of U.S. patent
application Ser. No. 10/330,696 filed Dec. 27, 2002, entitled
"Markers for Differential Diagnosis and Methods of Use Thereof,"
which claims priority to U.S. Provisional Patent Application No.
60/436,301 filed Dec. 24, 2002; and a continuation in part of U.S.
patent application Ser. No. 10/139,086 filed May 4, 2002, entitled
"Diagnostic Markers of Acute Coronary Syndromes and Methods of Use
Thereof," which claims priority to U.S. Provisional Patent
Application No. 60/315,642 filed Aug. 28, 2001; and a continuation
in part of International application PCT/US02/14219 filed May 4,
2002 which claims priority to U.S. Provisional Patent Application
No. 60/288,871 filed May 4, 2001 and U.S. Provisional Patent
Application No. 60/315,642 filed Aug. 28, 2001; and a continuation
in part of U.S. patent application Ser. No. 10/389,720 filed Mar.
13, 2003, entitled "Use of B-type Natriuretic Peptide as a
Prognostic Indicator in Acute Coronary Syndromes" which is a
divisional of U.S. patent application Ser. No. 09/835,298 filed
Apr. 13, 2001; and a continuation in part of International
application PCT/JUS02/11441 filed Apr. 11, 2002 which claims
priority to U.S. patent application Ser. No. 09/835,298 filed Apr.
13, 2001; and a continuation in part of U.S. patent application
Ser. No. 10/371,149 filed Feb. 20, 2003 entitled "Diagnostic
Markers of Stroke and Cerebral Injury and Methods of Use Thereof,"
which is a continuation in part of Ser. No. 10/225,082 filed Aug.
20, 2002, which claims priority to U.S. Provisional Patent
Application No. 60/313,775 filed Aug. 20, 2001; and a continuation
in part of International application PCT/US02/26604 filed Aug. 20,
2002 which claims priority to U.S. Provisional Patent Application
No. 60/313,775 filed Aug. 20, 2001, U.S. Provisional Patent
Application No. 60/346,485 filed Jan. 2, 2002, and to U.S.
Provisional Patent Application No. 60/334,964 filed Nov. 30, 2001;
and a continuation in part of U.S. patent application Ser. No.
10/331,127 filed Dec. 27, 2002 and Ser. No. 10/410,572 filed Apr.
8, 2003, both of which claim priority to U.S. Provisional Patent
Application No. 60/436,392 filed Dec. 24, 2002; a from each of
which priority is claimed, and each of which is hereby incorporated
by reference in its entirety, including all tables, figures, and
claims.
FIELD OF THE INVENTION
[0002] The present invention relates to the identification and use
of diagnostic markers for differential diagnosis of diseases. In a
various aspects, the invention relates to methods and compositions
able to determine the presence or absence of one, and preferably a
plurality, of diseases that exhibit one or more similar or
identical symptoms.
BACKGROUND OF THE INVENTION
[0003] The following discussion of the background of the invention
is merely provided to aid the reader in understanding the invention
and is not admitted to describe or constitute prior art to the
present invention.
[0004] The clinical presentation of certain diseases can often be
strikingly similar, even though the underlying diseases, and the
appropriate treatments to be given to one suffering from the
various diseases, can be completely distinct. For example, subjects
may present in an urgent care facility exhibiting a deceptively
simple constellation of apparent symptoms (e.g., fever, shortness
of breath, dizziness, headache) that may be characteristic of a
variety of unrelated conditions. Differential diagnosis methods
involve the comparison of symptoms and/or diagnostic test results
known to be associated with one or more diseases that exhibit a
similar clinical presentation to the symptoms and/or diagnostic
results exhibited by the subject, in order to identify the
underlying disease or condition present in the subject.
[0005] Taking shortness of breath (referred to clinically as
"dyspnea") as an example, patients often present in a clinical
setting with this symptom as the initial clinical presentation.
This symptom considered in isolation may be indicative of
conditions as diverse as asthma, chronic obstructive pulmonary
disease ("COPD"), tracheal stenosis, obstructive endobroncheal
tumor, pulmonary fibrosis, pneumoconiosis, lymphangitic
carcinomatosis, kyphoscoliosis, pleural effusion, amyotrophic
lateral sclerosis, congestive heart failure, coronary artery
disease, myocardial infarction, cardiomyopathy, valvular
dysfunction, left ventricle hypertrophy, pericarditis, arrhythmia,
pulmonary embolism, metabolic acidosis, chronic bronchitis,
pneumonia, anxiety, sepsis, aneurismic dissection, etc. See, e.g.,
Kelley's Textbook of Internal Medicine, 4.sup.th Ed., Lippincott
Williams & Wilkins, Philadelphia, Pa., 2000, pp. 2349-2354,
"Approach to the Patient With Dyspnea"; Mulrow et al., J. Gen. Int.
Med. 8: 383-92 (1993).
[0006] Differential diagnosis in the case of dyspnea involves
identifying the particular condition causing shortness of breath in
a given subject from amongst numerous possible causes. These
methods often require that the clinician integrate information
obtained from a battery of tests, leading to a clinical diagnosis
that most closely represents the range of symptoms and/or
diagnostic test results obtained for the subject. The tests
required may include radiography, electrocardiogram, exercise
treadmill testing, blood chemistry analysis, echocardiography,
bronchoprovocation testing, spirometry, pulse oximetry, esophageal
pH monitoring, laryngoscopy, computed tomography, histology,
cytology, magnetic resonance imaging, etc. See, e.g., Morgan and
Hodge, Am. Fam. Physician 57: 711-16 (1998). Because of the variety
of tests that may need to be performed, obtaining sufficient
information to arrive at a diagnosis can take hours or even
days.
[0007] Differential diagnosis of chest pain requires the clinician
to consider many possible causes, including differentiating between
respiratory pain and pain associated with angina, or myocardial
infarction and pleuritic and chest wall pain.
[0008] Differential diagnosis of diastolic and systolic dysfunction
in patients suffering from heart failure is important since the
therapies for each dysfunction are different. Further
differentiation of atrial fibrillation from heart failure is
critical for appropriate therapy.
[0009] In the area of infection, diffential diagnosis of viral
versus bacterial is critical to the clinician delivering the
appropriate therapy.
[0010] The acuteness or severity of the symptoms often dictates how
rapidly a diagnosis must be established and treatment initiated.
Immediate diagnosis and care of a patient experiencing a variety of
acute conditions associated with dyspnea and chest pain can be
critical. See, e.g., Harris, Aust. Fam. Physician 31: 802-06 (2002)
(asthma); Goldhaber, Eur. Respir. J. Suppl. 35: 22s-27s (2002)
(pulmonary embolism); Lundergan et al., Am. Heart J. 144: 456-62
(2002) (myocardial infarction). However, even in cases where the
apparent symptoms appear relatively stable, rapid diagnosis, and
the rapid initiation of treatment, can provide both relief from
immediate discomfort and advantageous improvement in prognosis.
[0011] Each reference cited in the preceeding section is hereby
incorporated by reference in its entirety, including all tables,
figures, and claims.
SUMMARY OF THE INVENTION
[0012] The present invention relates to the identification and use
of diagnostic markers for differential diagnosis of diseases. The
methods and compositions described herein can meet the need in the
art for rapid, sensitive and specific diagnostic assays to be used
in the diagnosis and differentiation of various diseases that are
related in terms of one or more clinical characteristics.
[0013] In various aspects, the invention relates to materials and
procedures for identifying the underlying cause of one or more
symptoms that, when considered in isolation, may be related to a
plurality of possible underlying diseases or conditions; to using
such markers in diagnosing and treating a patient and/or to monitor
the course of a treatment regimen; to using such markers to
identify subjects at risk for one or more adverse outcomes an
underlying disease or condition; and for screening compounds and
pharmaceutical compositions that might provide a benefit in
treating or preventing such diseases or conditions.
[0014] In a first aspect, the invention discloses methods for
determining the presence or absence of a disease in a subject that
is exhibiting a perceptible change in one or more physical
characteristics (that is, one or more "symptoms") that are
indicative of a plurality of possible etiologies underlying the
observed symptom(s). These methods comprise analyzing a test sample
obtained from the subject for the presence or amount of one or more
markers for one or more of the possible etiologies of the observed
symptom(s). The presence or amount of such marker(s) in a sample
obtained from the subject can be used to rule in or rule out one or
more of the possible etiologies, thereby either providing a
diagnosis (rule-in) and/or excluding one or more diagnoses
(rule-out).
[0015] In certain embodiments, these markers can be used to rule in
or rule out one or more possible etiologies of shortness of breath,
or "dyspnea." While the present invention is described hereinafter
generally in terms of the differential diagnosis of diseases
related to dyspnea, the skilled artisan will understand that the
concepts of symptom-based differential diagnosis described herein
are generally applicable to any physical characteristics that are
indicative of a plurality of possible etiologies such as fever,
chest pain (or "angina"), abdominal pain, neurologic dysfunction,
disturbances in metabolic state, such as aberrant water,
electrolyte, mineral, or acid-base metabolism, hypertension,
dizziness, headache, etc.
[0016] In preferred embodiments, the present invention relates to
methods in which a test sample is analyzed for the presence or
amount of a plurality of markers related to a plurality of possible
etiologies, so that the method is adapted to rule in or out a
plurality of possible underlying causes based upon the analysis of
a single sample. In the case of dyspnea, the plurality of markers
are preferably selected to rule in or out a plurality of the
following: asthma, atrial fibrillation, chronic obstructive
pulmonary disease ("COPD"), tracheal stenosis, obstructive
endobroncheal tumor, pulmonary fibrosis, pneumoconiosis,
lymphangitic carcinomatosis, kyphoscoliosis, pleural effusion,
amyotrophic lateral sclerosis, congestive heart failure, coronary
artery disease, myocardial infarction, cardiomyopathy, valvular
dysfunction, left ventricle hypertrophy, pericarditis, arrhythmia,
pulmonary embolism, metabolic acidosis, chronic bronchitis,
pneumonia, anxiety, sepsis, or aneurismic dissection. In a
particularly preferred embodiment, the methods relate to defining
the cause of dyspnea to rule in or rule out myocardial ischemia and
cardiac necrosis, heart failure and pulmonary embolism. In yet
another particularly preferred embodiment, the methods relate to
defining the cause of dyspnea to rule in or rule out myocardial
ischemia and cardiac necrosis, heart failure, pulmonary embolism
and atrial fibrillation.
[0017] In the case of abdominal pain, the plurality of markers are
preferably selected to rule in or out a plurality of the following:
aortic aneurysm, mesenteric embolism, pancreatitis, appendicitis,
myocardial infarction, one or more infectious diseases described
above, influenza, esophageal carcinoma, gastric adenocarcinoma,
colorectal adenocarcinoma, pancreatic tumors including ductal
adenocarcinoma, cystadenocarcinoma, and insulinoma.
[0018] In the case of disturbanes of metabolic state, the plurality
of markers are preferably selected to rule in or out a plurality of
the following: diabetes mellitus, diabetic ketoacidosis, alcoholic
ketoacidosis, respiratory acidosis, respiratory alkalosis,
nonketogenic hyperglycemia, hypoglycemia, renal failure,
interstitial renal disease, COPD, pneumonia, pulmonary and edema,
asthma.
[0019] In certain embodiments, etiologies other than a disease that
is to be ruled in or out, but which present similar symptoms, are
referred to herein as "mimics" of a disease. For example, marker(s)
able to differentiate one or more types of stroke from diseases
that present similar symptoms, but that are not stroke ("stroke
mimics"), are referred to herein as "stroke differential diagnostic
markers." The presence or amount of such marker(s) in a sample
obtained from the subject can be used to rule in or rule out one or
more of the following: stroke, thrombotic stroke, embolic stroke,
lacunar stroke, hypoperfusion, intracerebral hemorrhage, and
subarachnoid hemorrhage, thereby either providing a diagnosis
(rule-in) and/or excluding a diagnosis (rule-out). Similarly,
marker(s) able to differentiate congestive heart failure from
diseases that present similar symptoms, but that are not congestive
heart failure ("CHF mimics"), are referred to herein as "CHF
differential diagnostic markers;" marker(s) able to differentiate
myocardial infarction from diseases that present similar symptoms,
but that are not myocardial infarction ("MI mimics"), are referred
to herein as "MI differential diagnostic markers." In another
aspect, the present invention relates to methods and compositions
for further subdividing congestive heart failure by distinguishing
between systolic heart failure and diastolic heart failure. These
methods comprise analyzing a test sample obtained from the subject
for the presence or amount of one or more markers, the presence or
amount of which can be used to rule in or out systolic heart
failure and/or diastolic heart failure, or that can be used to
distinguish between these two causes of congestive heart failure.
Additional examples will be apparent to the skilled artisan from
the description provided herein.
[0020] In another aspect, the present invention relates to methods
and compositions for the differential diagnosis of atrial
fibrillation and heart failure. The methods comprise analyzing a
test sample obtained from the subject for the presence or amount of
one or more markers, the presence or amount of which can be used to
rule in or out heart failure or atrial fibrillation. In another
aspect of this embodiment, the methods can be used to distinguish
between systolic and diastolic dysfunction and atrial fibrillation.
In yet another aspect of this embodiment, the methods can be used
to distinguish between systolic and diastolic dysfunction, atrial
fibrillation, myocardial ischemia and cardiac necrosis.
[0021] In yet another aspect, the present invention relates to
methods and compositions for the differential diagnosis of aortic
dissection and myocardial ischemia and necrosis. The methods
comprise analyzing a test sample obtained from the subject for the
presence or amount of one or more markers, the presence or amount
of which can be used to rule in or out aortic dissection and
myocardial ischemia and cardiac necrosis. In another aspect of this
embodiment, the methods can be used to distinguish between aortic
dissection, myocardial ischemia and cardiac necrosis and heart
failure. In another aspect of this embodiment, the methods can be
used to distinguish between aortic dissection, myocardial ischemia
and cardiac necrosis, heart failure and atrial fibrillation.
[0022] Preferred markers of the invention can differentiate between
myocardial infarction, congestive heart failure, and pulmonary
embolism as a cause of dyspnea. Particularly preferred markers for
these diseases are cardiac-specific troponin isoforms, B-type
natriuretic peptide, and D-dimer, respectively. Each of these
preferred markers are described in detail hereinafter.
[0023] The markers described herein may be used individually, but
are preferably used as members of a marker "panel" comprising a
plurality of markers that are measured in a sample. Such a panel
may be analyzed in a number of fashions well known to those of
skill in the art. For example, each member of a panel may be
compared to a "normal" value, or a value identified as being
indicative of the presence or absence of a particular disease. A
particular diagnosis may depend upon the comparison of each marker
to this value; alternatively, if only a subset of markers are
outside of a normal range, this subset may be indicative of a
particular diagnosis. In preferred embodiments, markers and marker
panels are selected to exhibit at least 80% sensitivity, more
preferably at least 90% sensitivity, and even more preferably at
least 95% sensitivity, combined with at least 80% specificity, more
preferably at least 90% specificity, and even more preferably at
least 95% specificity. In particularly preferred embodiments, both
the sensitivity and specificity are at least 85%, more preferably
at least 90%, and even more preferably at least 95%.
[0024] Preferred marker panels of the present invention may be
correlated to the presence or absence of a plurality of
cardiovascular disorders. These marker panels preferably comprise
one or more markers related to blood pressure regulation, and one
or more markers related to cardiovascular damage. Additional
markers may be added to such a panel, including preferably one or
more markers related to inflammation and/or one or more markers
related to coagulation and hemostasis. Suitable markers for
inclusion in such panels are described in detail hereinafter.
[0025] Particularly preferred marker panels comprise, for example,
one or more first marker(s) selected from the group consisting of
atrial natriuretic factor, B-type natriuretic peptide or a marker
related to B-type natriuretic peptide, C-type natriuretic factor,
urotensin II, arginine vasopressin, aldosterone, angiotensin I,
angiotensin II, angiotensin III, bradykinin, calcitonin,
procalcitonin, calcitonin gene related peptide, adrenomedullin,
calcyphosine, endothelin-2, endothelin-3, rennin, ANP, and
urodilatin (referred to collectively as "markers related to blood
pressure regulation"); and one or more second markers selected from
the group consisting of free cardiac troponin I, free cardiac
troponin T, cardiac troponin I in a complex comprising one or both
of troponin T and troponin C, cardiac troponin T in a complex
comprising one or both of troponin I and troponin C, free and
complexed cardiac troponin I, free and complexed cardiac troponin
T, creatine kinase-MB, myoglobin, glycogen phosphorylase-BB,
annexin B, .beta.-enolase, heart-type fatty acid binding protein,
and S-100ao (referred to collectively as "markers related to
myocardial injury"). Additional markers related to blood pressure
regulation and myocardial injury are described hereinafter.
[0026] Additionally, one or more markers selected from the group
consisting of C-reactive protein, interleukins, interleukin-1
receptor agonist, CD54, CD106, monocyte chemotactic protein-1,
caspase-3, lipocalin-type prostaglandin D synthase, mast cell
tryptase, eosinophil cationic protein, KL-6, haptoglobin, tumor
necrosis factor a, tumor necrosis factor .beta., fibronectin, and
vascular endothelial growth factor ("VEGF") (referred to
collectively as "markers related to inflammation") may be included,
as well as markers selected from the group consisting of plasmin,
fibrinogen, D-dimer, .beta.-thromboglobulin, platelet factor 4,
fibrinopeptide A, platelet-derived growth factor, prothrombin
fragment 1+2, plasmin-.alpha.2-antiplasmin complex,
thrombin-antithrombin III complex, P-selectin, thrombin, von
Willebrand factor, tissue factor, and thrombus precursor protein
(referred to collectively as "markers related to coagulation and
hemostasis"). Additional markers related to inflammation and
markers related to coagulation and hemostasis are described
hereinafter.
[0027] Additional markers and/or marker classes may be added to
such panels to provide further ability to discriminate amongst
diseases. For example, one or more markers selected from the group
consisting of .alpha.-2 actin, basic calponin 1, .beta.-1 integrin,
acidic calponin, caldesmon, cysteine rich protein-2 ("CRP 2" or
"CSRP 2"), elastin, fibrillin 1, latent transforming growth factor
beta binding protein 4 ("LTBP 4"), smooth muscle myosin, smooth
muscle myosin heavy chain, and transgelin (referred to collectively
as "markers related to vascular tissue") may be included in such a
panel. Vascular tissue markers can in various embodiments be used
as markers of aortic dissection and/or peripheral vascular disease
or damage. Additional markers and marker classes are described
hereinafter.
[0028] These markers may be combined in various combinations. For
example, preferred panels may include B-type natriuretic peptide or
a marker related to B-type natriuretic peptide, creatine kinase-MB,
total cardiac troponin I, and myoglobin; B-type natriuretic peptide
or a marker related to B-type natriuretic peptide, creatine
kinase-MB, total cardiac troponin I, myoglobin, and C-reactive
protein; B-type natriuretic peptide or a marker related to B-type
natriuretic peptide, D-dimer, creatine kinase-MB, total cardiac
troponin I, and myoglobin; and/or B-type natriuretic peptide or a
marker related to B-type natriuretic peptide, D-dimer, creatine
kinase-MB, total cardiac troponin I, myoglobin, and C-reactive
protein. Such panels may distinguish a plurality of cardiovascular
disorders selected from the group consisting of myocardial
infarction, congestive heart failure, acute coronary syndrome,
unstable angina, and pulmonary embolism.
[0029] Likewise, a panel may comprise a plurality of markers
selected to diagnose and/or distinguish amongst a plurality of
cerebrovascular disorders. Preferred marker panels of the present
invention comprise one or more markers related to blood pressure
regulation, and one or more markers related to neural tissue
injury. Additional markers may be added to such a panel, including
preferably one or more markers related to inflammation, and/or one
or more markers related to apoptosis, and/or one or more acute
phase markers and/or one or more markers related to coagulation and
hemostasis.
[0030] Exemplary markers related to blood pressure regulation, to
inflammation, and to coagulation and hemostasis are described
above. One or more markers related to neural tissue injury include
those selected from the group consisting of precerebellin 1,
cerebillin 1., cerebellin 3, chimerin 1, chimerin 2, calbrain,
calbindin D, brain tubulin, brain fatty acid binding protein
("B-FABP"), brain derived neurotrophic factor ("BDNF"), carbonic
anhydrase XI, CACNA1A calcium channel gene, nerve growth factor
.beta., atrophin 1, apolipoprotein E4-1, protein 4.1B, 14-3-3
protein, ciliary neurotrophic factor, creatine kinase-BB, C-tau,
glial fibrillary acidic protein ("GFAP"), neural cell adhesion
molecule ("NCAM"), neuron specific enolase, S-100b, prostaglandin D
synthase, neurokinin A, neurotensin, and secretagogin. Additional
exemplary markers related to neural tissue injury are described
hereinafter. One or more markers related to apoptosis include those
selected from the group consisting of spectrin, cathepsin D,
caspase 3, s-acetyl glutathione, and ubiquitin fusion degradation
protein 1 homolog. Suitable additional markers for inclusion in
such panels are described in detail hereinafter. The presence or
amount of the markers in such panels may be correlated to the
presence or absence of a plurality of cerebrovascular
disorders.
[0031] Additionally, one or more acute phase reactants include
those selected from the group consisting of hepcidin, HSP-60,
HSP-65, HSP-70, S-FAS ligand, asymmetric dimethylarginine (an
endogenous inhibitor of nitric oxide synthase), matrix
metalloproteins 11, 3, and 9, defensin HBD 1, defensin HBD 2, serum
amyloid A, oxidized LDL, insulin like growth factor, transforming
growth factor .beta., e-selectin, glutathione-S-transferase,
hypoxia-inducible factor-1.alpha., inducible nitric oxide synthase
("I-NOS"), intracellular adhesion molecule, lactate dehydrogenase,
monocyte chemoattractant peptide-1 ("MCP-1"), n-acetyl aspartate,
prostaglandin E2, receptor activator of nuclear factor ("RANK")
ligand, TNF receptor superfamily member 1A, TNF.alpha., vascular
cell adhesion molecule, and cystatin C.
[0032] An exemplary marker panel selected to diagnose and/or
distinguish amongst a plurality of cerebrovascular disorders
includes a plurality (and preferably each) of B-type natriuretic
peptide or a marker related to B-type natriuretic peptide,
caspase-3, interleukin-8, creatine kinase-BB, C-reactive protein,
S-100b, matrix metalloprotein-9, and neural cell adhesion molecule.
The presence or amount of the markers in such panels may be
correlated to the presence or absence of a plurality of
cerebrovascular disorders. Additional markers and marker panels are
described hereinafter.
[0033] The presence or amount of the markers in such panels may be
correlated to the presence or absence of a plurality of
cerebrovascular disorders. Additional markers are described
hereinafter. As described hereinafter, the markers described herein
may be indicative of a plurality of diseases, depending on the
status of other markers in a panel. For example, certain markers
are generally elevated in inflammation resulting from a variety of
causes. Thus, alone, a single marker may not be diagnostic per se,
but as part of a panel, the marker can provide important diagnostic
and/or prognostic information.
[0034] In a related aspect, the presence or amount of markers that
are selected to diagnose and/or distinguish amongst a plurality of
cerebrovascular disorders may also be used prognostically, in order
to identify patients at risk for a future onset of a
cerebrovascular disorder. Such uses may find particular interest in
monitoring patients known to be at increased risk for such onset.
For example, patients undergoing carotid endarterectomy are known
to be at risk for cerebral ischemia. Outcomes of such ischemia
include intraoperative and perioperative stroke, neurologic
deficit, and death. Cerebral ischemia is also a risk of procedures
such as hypothermic circulatory arrest, aortic valve replacement,
mitral valve replacement, coronary artery surgery, endograft repair
of aortic aneurism, coronary artery bypass graft surgery, laryngeal
mask insertion, and repair of congenital heart defects. Thus, the
present invention also relates to methods and compositions for
monitoring the status of patients undergoing such procedures to
identify at-risk patients.
[0035] In another aspect, the present invention relates to methods
for identifying marker panels for use in the foregoing methods. The
sensitivity and specificity of a diagnostic test depends on more
than just the "quality" of the test--they also depend on the
definition of what constitutes an abnormal test. In practice,
Receiver Operating Characteristic curves, or "ROC" curves, are
typically calculated by plotting the value of a variable versus its
relative frequency in "normal" and "disease" populations. For any
particular marker, a distribution of marker levels for subjects
with and without a disease will likely overlap. Under such
conditions, a test does not absolutely distinguish normal from
disease with 100% accuracy, and the area of overlap indicates where
the test cannot distinguish normal from disease. A threshold is
selected, above which (or below which, depending on how a marker
moves with the disease) the test is considered to be abnormal and
below which the test is considered to be normal. The area under the
ROC curve is a measure of the probability that the perceived
measurement will allow correct identification of a condition. ROC
curves can be used even when test results don't necessarily give an
accurate numeric value for a marker level; that is, as long as one
can rank results, one can create an appropriate ROC curve. Such
methods are well known in the art. See, e.g., Hanley et al.,
Radiology 143: 29-36 (1982).
[0036] In preferred embodiments, particular thresholds for one or
more markers in a panel are not relied upon to determine if a
profile of marker levels obtained from a subject are indicative of
a particular diagnosis. Rather, the present invention may utilize
an evaluation of the entire profile of markers. By plotting ROC
curves for the sensitivity of a particular panel of markers versus
1-(specificity) for the panel at various cutoffs, a profile of
marker measurements from a subject may be considered together to
provide a global probability (expressed either as a numeric score
or as a percentage risk) that the symptom(s) observed in an
individual are caused by a partiuclar underlying disease. In such
embodiments, an increase in a certain subset of markers may be
sufficient to indicate a particular diagnosis in one patient, while
an increase in a different subset of markers may be sufficient to
indicate the same or a different diagnosis in another patient.
[0037] One or more markers may lack predictive value when
considered alone, but when used as part of a panel, such markers
may be of great value in determining a particular diagnosis.
Weighting factors may also be applied to one or more markers in a
panel, for example, when a marker is of particularly high utility
in identifying a particular diagnosis. While the exemplary panels
described herein can provide the ability to determine a diagnosis
underlying, e.g., dyspnea, one or more markers may be replaced,
added, or subtracted from these exemplary panels while still
providing clinically useful results.
[0038] In yet other embodiments, multiple determinations of one or
more markers can be made, and a temporal change in the markers can
be used to rule in or out one or more particular etiologies for
observed symptom(s). For example, one or more markers may be
determined at an initial time, and again at a second time, and the
change (or lack thereof) in the marker level(s) over time
determined. In such embodiments, an increase in the marker from the
initial time to the second time may be diagnostic of a particular
disease underlying one or more symptoms. Likewise, a decrease in
the marker from the initial time to the second time may be
indicative of a particular disease underlying one or more symptoms.
Temporal changes in one or more markers may also be used together
with single time point marker levels to increase the discriminating
power of marker panels.
[0039] In yet another embodiment, multiple determinations of one or
more diagnostic or prognostic markers can be made, and a temporal
change in the marker can be used to monitor the efficacy
appropriate therapies. In such an embodiment, one might expect to
see a decrease or an increase in the marker(s) over time during the
course of effective therapy.
[0040] The skilled artisan will understand that, while in certain
embodiments comparative measurements are made of the same
diagnostic marker at multiple time points, one could also measure a
given marker at one time point, and a second marker at a second
time point, and a comparison of these markers may provide
diagnostic information. Similarly, the skilled artisan will
understand that serial measurements and changes in markers or the
combined result over time may also be of diagnostic and/or
prognostic value.
[0041] The skilled artisan will understand that associating one or
more diagnostic markers with the presence or absence of a
particular disease is a statistical analysis. For example, the
presence or absence of a particular marker level may signal that a
patient is more likely to suffer from a disease, as determined by a
level of statistical significance. Statistical significance is
often determined by comparing two or more populations, and
determining a confidence interval and/or a p value. See, e.g.,
Dowdy and Wearden, Statistics for Research, John Wiley & Sons,
New York, 1983. Preferred confidence intervals of the invention are
90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9% and 99.99%, while preferred
p values are 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, and
0.0001.
[0042] In yet another aspect, the invention relates to methods for
determining a treatment regimen for use in a subject. The methods
preferably comprise identifying the underlying cause of one or more
symptoms that, when considered in isolation, may be related to a
plurality of possible underlying diseases or conditions as
described herein. One or more treatment regimens can then be
selected to treat the underlying disease in the subject.
[0043] In a related aspect, the invention relates to methods for
determining a treatment regimen for use in a subject suffering from
congestive heart failure. The methods preferably comprise
performing the methods described herein to rule in or out systolic
heart failure and/or diastolic heart failure, or to distinguish
between these two causes of congestive heart failure. One or more
treatment regimens can then be selected to treat the type of
congestive heart failure in the subject.
[0044] In a further aspect, the invention relates to kits for
identifying the underlying cause of one or more symptoms that, when
considered in isolation, may be related to a plurality of possible
underlying diseases or conditions. These kits preferably comprise
devices and reagents for measuring one or more marker levels in a
patient sample, and instructions for performing the assay.
Optionally, the kits may contain one or more means for correlating
marker level(s) in order to rule in or rule out one or more
potential etiologies of the observed symptoms. Such kits preferably
contain sufficient reagents to perform one or more such
determinations, and/or Food and Drug Administration (FDA)-approved
labeling.
[0045] In a related aspect, the invention relates to kits to rule
in or out systolic heart failure and/or diastolic heart failure, or
to distinguish between these two causes of congestive heart
failure. These kits preferably comprise devices and reagents for
measuring one or more marker levels in a patient sample, and
instructions for performing the assay. Optionally, the kits may
contain one or more means for correlating marker level(s) in order
to distinguish between systolic heart failure and diastolic heart
failure. Such kits preferably contain sufficient reagents to
perform one or more such determinations, and/or Food and Drug
Administration (FDA)-approved labeling.
[0046] In yet a further aspect, the invention relates to devices
for identifying the underlying cause of one or more symptoms that,
when considered in isolation, may be related to a plurality of
possible underlying diseases or conditions. Such devices preferably
contain a plurality of discrete, independently addressable
locations, or "diagnostic zones," each of which is related to a
particular marker of interest. Following reaction of a sample with
the devices, a signal is generated from the diagnostic zone(s),
which may then be correlated to the presence or amount of the
markers of interest. Such markers may then be used to rule in or
out one or more potential etiologies of the observed symptoms.
[0047] In a related aspect, the invention relates to devices to
rule in or out systolic heart failure and/or diastolic heart
failure, or to distinguish between these two causes of congestive
heart failure. Such devices preferably contain a plurality of
diagnostic zones, each of which is related to a particular marker
of interest. Following reaction of a sample with the devices, a
signal is generated from the diagnostic zone(s), which may then be
correlated to the presence or amount of the markers of interest.
Such markers may then be used to distinguish between systolic heart
failure and diastolic heart failure.
DETAILED DESCRIPTION OF THE INVENTION
[0048] The present invention relates to methods and compositions
for symptom-based differential diagnosis of diseases in
subjects.
[0049] Patients presenting for medical treatment often exhibit one
or a few primary observable changes in bodily characteristics or
functions that are indicative of disease. Often, these "symptoms"
are nonspecific, in that a number of potential diseases can present
the same observable symptom or symptoms. A typical list of
nonspecific symptoms might include one or more of the following:
shortness of breath (or dyspnea), chest pain, fever, dizziness, and
headache. These symptoms can be quite common, and the number of
diseases that must be considered by the clinician can be
astoundingly broad.
[0050] Taking shortness of breath (referred to clinically as
"dyspnea") as an example, this symptom considered in isolation may
be indicative of conditions as diverse as asthma, chronic
obstructive pulmonary disease ("COPD"), tracheal stenosis,
obstructive endobroncheal tumor, pulmonary fibrosis,
pneumoconiosis, lymphangitic carcinomatosis, kyphoscoliosis,
pleural effusion, amyotrophic lateral sclerosis, congestive heart
failure, coronary artery disease, myocardial infarction, atrial
fibrillation, cardiomyopathy, valvular dysfunction, left ventricle
hypertrophy, pericarditis, arrhythmia, pulmonary embolism,
metabolic acidosis, chronic bronchitis, pneumonia, anxiety, sepsis,
aneurismic dissection, etc. See, e.g., Kelley's Textbook of
Internal Medicine, 4.sup.th Ed., Lippincott Williams & Wilkins,
Philadelphia, Pa., 2000, pp. 2349-2354, "Approach to the Patient
With Dyspnea"; Mulrow et al., J. Gen. Int. Med. 8: 383-92
(1993).
[0051] Similarly, chest pain, when considered in isolation, may be
indicative of stable angina, unstable angina, myocardial ischemia,
atrial fibrillation, myocardial infarction, musculoskeletal injury,
cholecystitis, gastroesophageal reflux, pulmonary embolism,
pericarditis, aortic dissection, pneumonia, anxiety, etc. Moreover,
the classification of chest pain as stable or unstable angina (or
even mild myocardial infarction) in cases other than definitive
myocardial infarction is completely subjective. The diagnosis, and
in this case the distinction, is made not by angiography, which may
quantify the degree of arterial occlusion, but rather by a
physician's interpretation of clinical symptoms.
[0052] Differential diagnosis refers to methods for diagnosing the
particular disease(s) underlying the symptoms in a particular
subject, based on a comparison of the characteristic features
observable from the subject to the characteristic features of those
potential diseases. Depending on the breadth of diseases that must
be considered in the differential diagnosis, the types and number
of tests that must be ordered by a clinician can be quite large. In
the case of dyspnea for example, the clinician may order tests from
a group that includes radiography, electrocardiogram, exercise
treadmill testing, blood chemistry analysis, echocardiography,
bronchoprovocation testing, spirometry, pulse oximetry, esophageal
pH monitoring, laryngoscopy, computed tomography, histology,
cytology, magnetic resonance imaging, etc. See, e.g., Morgan and
Hodge, Am. Fam. Physician 57: 711-16 (1998). The clinician must
then integrate information obtained from a battery of tests,
leading to a clinical diagnosis that most closely represents the
range of symptoms and/or diagnostic test results obtained for the
subject.
[0053] The present invention describes methods and compositions
that can assist in the differential diagnosis of one or more
nonspecific symptoms by providing diagnostic markers that are
designed to rule in or out one, and preferably a plurality, of
possible etiologies for the observed symptoms. The concept of
symptom-based differential diagnosis described herein can provide
panels of diagnostic markers designed be considered in concert to
distinguish between possible diseases that underlie a nonspecific
symptom observed in a patient.
[0054] Definitions
[0055] The term "fever" as used herein refers to a body temperature
greater than 100.degree. C. orally or 100.8.degree. C. rectally. In
the case of fever, a plurality of markers are preferably selected
to rule in or out a plurality of the following: sepsis; arteritis;
sarcoidosis; and one or more infectious diseases, including
infection by Staphyloccus species, Nisseria species, Pneumococcal
species, Listeria species, Anthrax, Nocardia species, Salmonella
species, Shigella species, Haemophilus species, Brucella species,
Vibrio species including V. cholerae, Franciscella tularensis,
Yersinia pestis, Pseudomonas species, Clostridia species including
C. tetani, C. perfringens, C. ramosum, C. botulinum, and C.
septicum, Actinomyces species, Treponema pallidum, Borrelia species
including B. burgdorferi, Leptospira species, Mycobacterium species
including M. tuberculosis, M. bovis, M. leprae, and M. africanum,
Histoplasma species, Escherichia coli, Coccidioides species,
Blastomyces species, Paracoccidioides species, Sporothrix species,
Cryptococcus species, Candida species, and Aspergillus species;
Rickettsial diseases including Rocky Mountain spotted fever, Q
fever, typhus, trench fever, and cat-scratch fever; parasitic
diseases including Malaria, Babesiosis, African sleeping sickness,
Trypanosomiasis, Leishmaniasis, Toxoplasmosis, and Amebiasis; viral
infection by influenza virus, parainfluenza virus, mumps virus,
adenovirus, respiratory syncytial virus, rhinovirus, poliovirus,
coxackievirus, echovirus, rubeola virus, rubella virus, parvovirus,
hepatitis A, B, C, D, or E, cytomegalovirus, Epstein-Barr virus,
Herpes simplex virus, Varicella-zoster virus, Alphavirus,
Flaviviruses including yellow fever virus, dengue fever virus,
Japanese encephalitis virus, and St. Louis encephalitis virus, West
Nile virus, Colorado tick fever virus, Rabies virus, Arenavirus,
Marburg agent, and Ebola virus.
[0056] The term "cerebrovascular disorder" as used herein refers to
vascular and parenchymal cerebral abnormalities. The term is
intended to include ischemic stroke (e.g., atherogenic embolism,
thrombotic occlusion, lacunar stroke, cardioembolytic stroke),
hemorrhagic stroke, transient ischemic attack, subarachnoid
hemorrhage, cerebral vasospasm, hypertensive small vessel disease,
vascular inflammatory conditions, aneurysm, arteriovenous
malformations, and cerebral amyloid angiopathy (a disorder in which
deposition of amyloid within the arterial media and adventitia
leads to intracerebral hemorrhage). This list is not meant to be
limiting.
[0057] The term "cardiovascular disorder" as used herein refers to
abnormalities of the heart and vasculature. The term is intended to
include, but is not limited to, renovascular hypertension,
congestive heart failure, aortic aneurysm, iliac or femoral
aneurysm, pulmonary embolism, myocardial infarction, acute coronary
syndrome, angina, primary hypertension, atrial fibrillation,
systolic dysfunction, diastolic dysfunction, myocarditis,
atherosclerosis, atrial tachycardia, ventricular fibrillation,
endocarditis, and peripheral vascular disease.
[0058] The term "neurologic dysfunction" as used herein refers to a
loss of one or more normal physiological or mental functions having
a neurogenic etiology. The skilled artisan will understand that
neurologic dysfunction is a common symptom in various systemic
disorders (e.g., alcoholism, vascular disease, stroke,
autoimmunity, metabolic disorders, aging, etc.). Specific
neurologic dysfunctions include, but are not limited to, pain,
headache, aphasia, apraxia, agnosia, amnesia, stupor, coma,
delirium, dementia, seizure, migraine insomnia, hypersomnia, sleep
apnea, tremor, dyskinesia, paralysis, etc.
[0059] The term "hypertension" as used herein refers to a systolic
blood pressure of greater than or equal to 140 mm Hg and/or a
diastolic blood pressure of greater than or equal to 90 mm Hg.
Hypertension can include isolated systolic hypertension (i.e., no
elevation in diastolic blood pressure). In the case of
hypertension, the plurality of markers are preferably selected to
rule in or out a plurality of the following: left ventricular
failure, atherosclerosis, renal disease including chronic
glomerulonephritis, and polycystic renal disease, coartation of the
aorta, renal arteriall stenosis, and hyperparathyroidism.
[0060] The term "condition within the differential diagnosis of a
symptom" as used herein refers to a pathologic state that is known
to be causative of a particular perceptible change in one or more
physical characteristics exhibited by a subject suffering from the
pathologic state, as compared to a normal subject. The concept of
differential diagnosis is well established to those of skill in the
art. See, e.g., Beck, Tutorials in Differential Diagnosis,
Churchill Livingstone, 2002; Zackon, Pulmonary Differential
Diagnosis, Elsevier, 2000; Jamison, Differential Diagnosis for
Primary Practice, Churchill Livingstone, 1999; Bouchier et al.,
French's Index of Differential Diagnosis, Oxford University Press,
1997.
[0061] The term "marker" as used herein refers to proteins,
polypeptides, phospholipids, or small molecules to be used as
targets for screening test samples obtained from subjects.
"Proteins or polypeptides" used as markers in the present invention
are contemplated to include any fragments thereof, in particular,
immunologically detectable fragments.
[0062] The term "related marker" as used herein refers to one or
more fragments of a particular marker or its biosynthetic parent
that may be detected as a surrogate for the marker itself or as
independent markers. For example, human BNP is derived by
proteolysis of a 108 amino acid precursor molecule, referred to
hereinafter as BNP.sub.1-108. Mature BNP, or "the BNP natriuretic
peptide," or "BNP-32" is a 32 amino acid molecule representing
amino acids 77-108 of this precursor, which may be referred to as
BNP.sub.77-108. The remaining residues 1-76 are referred to
hereinafter as BNP.sub.1-76.
[0063] The sequence of the 108 amino acid BNP precursor pro-BNP
(BNP.sub.1-108) is as follows, with mature BNP (BNP.sub.77-108)
underlined:
1 HPLGSPGSAS DLETSGLQEQ RNHLQGKLSE LQVEQTSLEP LQESPRPTGV 50 (SEQ ID
NO: 1) WKSREVATEG IRGHRKMVLY TLRAPRSPKM VQGSGCFGRK MDRISSSSGL 100
GCKVLRRH. 108
[0064] BNP.sub.1-108 is synthesized as a larger precursor
pre-pro-BNP having the following sequence (with the "pre" sequence
shown in bold):
2 MDPQTAPSRA LLLLLFLHLA FLGGRSHPLG SPGSASDLET SGLQEQRNHL 50 (SEQ ID
NO: 2) QGKLSELQVE QTSLEPLQES PRPTGVWKSR EVATEGIRGH RKMVLYTLRA 100
PRSPKMVQGS GCFGRKMDRI SSSSGLGCKV LRRH. 134
[0065] While mature BNP itself may be used as a marker in the
present invention, the prepro-BNP, BNP.sub.1-108 and BNP.sub.1-76
molecules represent BNP-related markers that may be measured either
as surrogates for mature BNP or as markers in and of themselves. In
addition, one or more fragments of these molecules, including
BNP-related polypeptides selected from the group consisting of
BNP.sub.77-106, BNP.sub.79-106, BNP.sub.76-107, BNP.sub.69-108,
BNP.sub.79-108, BNP.sub.80-108, BNP.sub.81-108, BNP.sub.83-108,
BNP.sub.39-86, BNP.sub.53-85, BNP.sub.66-98, BNP.sub.30-103,
BNP.sub.1-107, BNP.sub.9-106, and BNP.sub.3-108 may also be present
in circulation. In addition, natriuretic peptide fragments,
including BNP fragments, may comprise one or more oxidizable
methionines, the oxidation of which to methionine sulfoxide or
methionine sulfone produces additional BNP-related markers. See,
e.g., U.S. patent Ser. No. 10/419,059, filed Apr. 17, 2003, which
is hereby incorporated by reference in its entirety including all
tables, figures and claims.
[0066] Because production of BNP-related markers is an ongoing
process that may be a function of, inter alia, the elapsed time
between onset of an event triggering natriuretic peptide release
into the tissues and the time the sample is obtained or analyzed;
the elapsed time between sample acquisition and the time the sample
is analyzed; the type of tissue sample at issue; the storage
conditions; the quantity of proteolytic enzymes present; etc., it
may be necessary to consider this degradation when both designing
an assay for one or more natriuretic peptides, and when performing
such an assay, in order to provide an accurate prognostic or
diagnostic result. In addition, individual antibodies that
distinguish amongst a plurality of natriuretic peptide (e.g., BNP)
fragments may be individually employed to separately detect the
presence or amount of different fragments. The results of this
individual detection may provide a more accurate prognostic or
diagnostic result than detecting the plurality of fragments in a
single assay. For example, different weighting factors may be
applied to the various fragment measurements to provide a more
accurate estimate of the amount of natriuretic peptide originally
present in the sample.
[0067] In a similar fashion, many of the markers described herein
are synthesized as larger precursor molecules, which are then
processed to provide mature marker; and/or are present in
circulation in the form of fragments of the marker. Thus, "related
markers" to each of the markers described herein may be identified
and used in an analogous fashion to that described above for
BNP.
[0068] Preferably, the methods described hereinafter utilize one or
more markers that are derived from the subject. The term
"subject-derived marker" as used herein refers to protein,
polypeptide, phospholipid, nucleic acid, prion, or small molecule
markers that are expressed or produced by one or more cells of the
subject. The presence, absence, or amount of one or more markers
may indicate that a particular disease is present, or may indicate
that a particular disease is absent. Additional markers may be used
that are derived not from the subject, but rather that are
expressed by pathogenic or infectious organisms that are correlated
with a particular disease. Such markers are preferably protein,
polypeptide, phospholipid, nucleic acid, prion, or small molecule
markers that identify the infectious diseases described above.
[0069] The term "test sample" as used herein refers to a sample of
bodily fluid obtained for the purpose of diagnosis, prognosis, or
evaluation of a subject of interest, such as a patient. In certain
embodiments, such a sample may be obtained for the purpose of
determining the outcome of an ongoing condition or the effect of a
treatment regimen on a condition. Preferred test samples include
blood, serum, plasma, cerebrospinal fluid, urine, saliva, sputum,
and pleural effusions. In addition, one of skill in the art would
realize that some test samples would be more readily analyzed
following a fractionation or purification procedure, for example,
separation of whole blood into serum or plasma components.
[0070] As used herein, a "plurality" as used herein refers to at
least two. Preferably, a plurality refers to at least 3, more
preferably at least 5, even more preferably at least 10, even more
preferably at least 15, and most preferably at least 20. In
particularly preferred embodiments, a plurality is a large number,
i.e., at least 100.
[0071] The term "subject" as used herein refers to a human or
non-human organism. Thus, the methods and compositions described
herein are applicable to both human and veterinary disease.
Further, while a subject is preferably a living organism, the
invention described herein may be used in post-mortem analysis as
well. Preferred subjects are "patients," i.e., living humans that
are receiving medical care. This includes persons with no defined
illness who are being investigated for signs of pathology.
[0072] The term "diagnosis" as used herein refers to methods by
which the skilled artisan can estimate and/or determine whether or
not a patient is suffering from a given disease or condition. The
skilled artisan often makes a diagnosis on the basis of one or more
diagnostic indicators, i.e., a marker, the presence, absence, or
amount of which is indicative of the presence, severity, or absence
of the condition.
[0073] Similarly, a prognosis is often determined by examining one
or more "prognostic indicators." These are markers, the presence or
amount of which in a patient (or a sample obtained from the
patient) signal a probability that a given course or outcome will
occur. For example, when one or more prognostic indicators reach a
sufficiently high level in samples obtained from such patients, the
level may signal that the patient is at an increased probability
for experiencing a future stroke in comparison to a similar patient
exhibiting a lower marker level. A level or a change in level of a
prognostic indicator, which in turn is associated with an increased
probability of morbidity or death, is referred to as being
"associated with an increased predisposition to an adverse outcome"
in a patient. Preferred prognostic markers can predict the onset of
delayed neurologic deficits in a patient after stroke, or the
chance of future stroke.
[0074] The term "correlating," as used herein in reference to the
use of diagnostic and markers, refers to comparing the presence or
amount of the marker(s) in a patient to its presence or amount in
persons known to suffer from, or known to be at risk of, a given
condition; or in persons known to be free of a given condition. As
discussed above, a marker level in a patient sample can be compared
to a level known to be associated with a specific diagnosis. The
sample's marker level is said to have been correlated with a
diagnosis; that is, the skilled artisan can use the marker level to
determine whether the patient suffers from a specific type
diagnosis, and respond accordingly. Alternatively, the sample's
marker level can be compared to a marker level known to be
associated with a good outcome (e.g., the absence of disease,
etc.). In preferred embodiments, a profile of marker levels are
correlated to a global probability or a particular outcome using
ROC curves.
[0075] The phrase "determining the diagnosis" as used herein refers
to methods by which the skilled artisan can determine the presence
or absence of a particular disease in a patient. The term
"diagnosis" does not refer to the ability to determine the presence
or absence of a particular disease with 100% accuracy, or even that
a given course or outcome is more likely to occur than not.
Instead, the skilled artisan will understand that the term
"diagnosis" refers to an increased probability that a certain
disease is present in the subject. In preferred embodiments, a
diagnosis indicates about a 5% increased chance that a disease is
present, about a 10% chance, about a 15% chance, about a 20%
chance, about a 25% chance, about a 30% chance, about a 40% chance,
about a 50% chance, about a 60% chance, about a 75% chance, about a
90% chance, and about a 95% chance. The term "about" in this
context refers to +/-2%.
[0076] The term "discrete" as used herein refers to areas of a
surface that are non-contiguous. That is, two areas are discrete
from one another if a border that is not part of either area
completely surrounds each of the two areas.
[0077] The term "independently addressable" as used herein refers
to discrete areas of a surface from which a specific signal may be
obtained.
[0078] The term "antibody" as used herein refers to a peptide or
polypeptide derived from, modeled after or substantially encoded by
an immunoglobulin gene or immunoglobulin genes, or fragments
thereof, capable of specifically binding an antigen or epitope.
See, e.g. Fundamental Immunology, 3.sup.rd Edition, W. E. Paul,
ed., Raven Press, N.Y. (1993); Wilson (1994) J. Immunol. Methods
175:267-273; Yarmush (1992) J. Biochem. Biophys. Methods 25:85-97.
The term antibody includes antigen-binding portions, i.e., "antigen
binding sites," (e.g., fragments, subsequences, complementarity
determining regions (CDRs)) that retain capacity to bind antigen,
including (i) a Fab fragment, a monovalent fragment consisting of
the VL, VH, CL and CH1 domains; (ii) a F(ab').sub.2 fragment, a
bivalent fragment comprising two Fab fragments linked by a
disulfide bridge at the hinge region; (iii) a Fd fragment
consisting of the VH and CH1 domains; (iv) a Fv fragment consisting
of the VL and VH domains of a single arm of an antibody, (v) a dAb
fragment (Ward et al., (1989) Nature 341:544-546), which consists
of a VH domain; and (vi) an isolated complementarity determining
region (CDR). Single chain antibodies are also included by
reference in the term "antibody."
[0079] Identification of Marker Panels
[0080] In accordance with the present invention, there are provided
methods and systems for the identification of one or more markers
for the differential diagnosis of one or more nonspecific symptoms
exhibited by a subject. Suitable methods for identifying markers
useful for the diagnosis of disease states are described in detail
in U.S. Provisional Patent Application No. 60/436,392 filed Dec.
24, 2002, entitled METHOD AND SYSTEM FOR DISEASE DETECTION USING
MARKER COMBINATIONS, and U.S. patent application Ser. No.
10/331,127 filed Dec. 27, 2002, entitled METHOD AND SYSTEM FOR
DISEASE DETECTION USING MARKER COMBINATIONS, each of which is
hereby incorporated by reference in its entirety, including all
tables, figures, and claims.
[0081] One skilled in the art will also recognize that univariate
analysis of markers can be performed and the data from the
univariate analyses of multiple markers can be combined to form
panels of markers to differentiate different disease conditions. In
forming panels of markers to define the cause of dypsnea, for
example, markers related to each cause of dypsnea should be
considered.
[0082] In developing a panel of markers useful in differential
diagnosis, data for a number of potential markers may be obtained
from a group of subjects by testing for the presence or level of
certain markers. The group of subjects is divided into two sets.
The first set includes subjects who have been confirmed as having a
disease or, more generally, being in a first condition state. For
example, this first set of patients may be those that have recently
had a stroke. The confirmation of this condition state may be made
through a more rigorous and/or expensive testing confirm the
condition state. Hereinafter, subjects in this first set will be
referred to as "diseased".
[0083] The second set of subjects is simply those who do not fall
within the first set. Subjects in this second set will hereinafter
be referred to as "non-diseased". Preferably, the first set and the
second set each have an approximately equal number of subjects.
[0084] The data obtained from subjects in these sets includes
levels of a plurality of markers. Preferably, data for the same set
of markers is available for each patient. This set of markers may
include all candidate markers that may be suspected as being
relevant to the detection of a particular disease or condition.
Actual known relevance is not required. Embodiments of the methods
and systems described herein may be used to determine which of the
candidate markers are most relevant to the diagnosis of the disease
or condition. The levels of each marker in the two sets of subjects
may be distributed across a broad range, e.g., as a Gaussian
distribution. However, no distribution fit is required.
[0085] As noted above, a marker often is incapable of definitively
identifying a patient as either diseased or non-diseased. For
example, if a patient is measured as having a marker level that
falls within the overlapping region, the results of the test will
be useless in diagnosing the patient. An artificial cutoff may be
used to distinguish between a positive and a negative test result
for the detection of the disease or condition. Regardless of where
the cutoff is selected, the effectiveness of the single marker as a
diagnosis tool is unaffected. Changing the cutoff merely trades off
between the number of false positives and the number of false
negatives resulting from the use of the single marker. The
effectiveness of a test having such an overlap is often expressed
using a ROC (Receiver Operating Characteristic) curve. ROC curves
are well known to those skilled in the art.
[0086] The horizontal axis of the ROC curve represents
(1-specificity), which increases with the rate of false positives.
The vertical axis of the curve represents sensitivity, which
increases with the rate of true positives. Thus, for a particular
cutoff selected, the value of (1-specificity) may be determined,
and a corresponding sensitivity may be obtained. The area under the
ROC curve is a measure of the probability that the measured marker
level will allow correct identification of a disease or condition.
Thus, the area under the ROC curve can be used to determine the
effectiveness of the test.
[0087] As discussed above, the measurement of the level of a single
marker may have limited usefulness. The measurement of additional
markers provides additional information, but the difficulty lies in
properly combining the levels of two potentially unrelated
measurements. In the methods and systems according to embodiments
of the present invention, data relating to levels of various
markers for the sets of diseased and non-diseased patients may be
used to develop a panel of markers to provide a useful panel
response. The data may be provided in a database such as Microsoft
Access, Oracle, other SQL databases or simply in a data file. The
database or data file may contain, for example, a patient
identifier such as a name or number, the levels of the various
markers present, and whether the patient is diseased or
non-diseased.
[0088] Next, an artificial cutoff region may be initially selected
for each marker. The location of the cutoff region may initially be
selected at any point, but the selection may affect the
optimization process described below. In this regard, selection
near a suspected optimal location may facilitate faster convergence
of the optimizer. In a preferred method, the cutoff region is
initially centered about the center of the overlap region of the
two sets of patients. In one embodiment, the cutoff region may
simply be a cutoff point. In other embodiments, the cutoff region
may have a length of greater than zero. In this regard, the cutoff
region may be defined by a center value and a magnitude of length.
In practice, the initial selection of the limits of the cutoff
region may be determined according to a pre-selected percentile of
each set of subjects. For example, a point above which a
pre-selected percentile of diseased patients are measured may be
used as the right (upper) end of the cutoff range.
[0089] Each marker value for each patient may then be mapped to an
indicator. The indicator is assigned one value below the cutoff
region and another value above the cutoff region. For example, if a
marker generally has a lower value for non-diseased patients and a
higher value for diseased patients, a zero indicator will be
assigned to a low value for a particular marker, indicating a
potentially low likelihood of a positive diagnosis. In other
embodiments, the indicator may be calculated based on a polynomial.
The coefficients of the polynomial may be determined based on the
distributions of the marker values among the diseased and
non-diseased subjects.
[0090] The relative importance of the various markers may be
indicated by a weighting factor. The weighting factor may initially
be assigned as a coefficient for each marker. As with the cutoff
region, the initial selection of the weighting factor may be
selected at any acceptable value, but the selection may affect the
optimization process. In this regard, selection near a suspected
optimal location may facilitate faster convergence of the
optimizer. In a preferred method, acceptable weighting coefficients
may range between zero and one, and an initial weighting
coefficient for each marker may be assigned as 0.5. In a preferred
embodiment, the initial weighting coefficient for each marker may
be associated with the effectiveness of that marker by itself. For
example, a ROC curve may be generated for the single marker, and
the area under the ROC curve may be used as the initial weighting
coefficient for that marker.
[0091] Next, a panel response may be calculated for each subject in
each of the two sets. The panel response is a function of the
indicators to which each marker level is mapped and the weighting
coefficients for each marker. In a preferred embodiment, the panel
response (R) for a each subject 0) is expressed as:
R.sub.j=.SIGMA.w.sub.iI.sub.i,j,
[0092] where i is the marker index, j is the subject index, w.sub.i
is the weighting coefficient for marker i, I is the indicator value
to which the marker level for marker i is mapped for subject j, and
.SIGMA. is the summation over all candidate markers i.
[0093] One advantage of using an indicator value rather than the
marker value is that an extraordinarily high or low marker levels
do not change the probability of a diagnosis of diseased or
non-diseased for that particular marker. Typically, a marker value
above a certain level generally indicates a certain condition
state. Marker values above that level indicate the condition state
with the same certainty. Thus, an extraordinarily high marker value
may not indicate an extraordinarily high probability of that
condition state. The use of an indicator which is constant on one
side of the cutoff region eliminates this concern.
[0094] The panel response may also be a general function of several
parameters including the marker levels and other factors including,
for example, race and gender of the patient. Other factors
contributing to the panel response may include the slope of the
value of a particular marker over time. For example, a patient may
be measured when first arriving at the hospital for a particular
marker. The same marker may be measured again an hour later, and
the level of change may be reflected in the panel response.
Further, additional markers may be derived from other markers and
may contribute to the value of the panel response. For example, the
ratio of values of two markers may be a factor in calculating the
panel response.
[0095] Having obtained panel responses for each subject in each set
of subjects, the distribution of the panel responses for each set
may now be analyzed. An objective function may be defined to
facilitate the selection of an effective panel. The objective
function should generally be indicative of the effectiveness of the
panel, as may be expressed by, for example, overlap of the panel
responses of the diseased set of subjects and the panel responses
of the non-diseased set of subjects. In this manner, the objective
function may be optimized to maximize the effectiveness of the
panel by, for example, minimizing the overlap.
[0096] In a preferred embodiment, the ROC curve representing the
panel responses of the two sets of subjects may be used to define
the objective function. For example, the objective function may
reflect the area under the ROC curve. By maximizing the area under
the curve, one may maximize the effectiveness of the panel of
markers. In other embodiments, other features of the ROC curve may
be used to define the objective function. For example, the point at
which the slope of the ROC curve is equal to one may be a useful
feature. In other embodiments, the point at which the product of
sensitivity and specificity is a maximum, sometimes referred to as
the "knee," may be used. In an embodiment, the sensitivity at the
knee may be maximized. In further embodiments, the sensitivity at a
predetermined specificity level may be used to define the objective
function. Other embodiments may use the specificity at a
predetermined sensitivity level may be used. In still other
embodiments, combinations of two or more of these ROC-curve
features may be used.
[0097] It is possible that one of the markers in the panel is
specific to the disease or condition being diagnosed. When such
markers are present at above or below a certain threshold, the
panel response may be set to return a "positive" test result. When
the threshold is not satisfied, however, the levels of the marker
may nevertheless be used as possible contributors to the objective
function.
[0098] An optimization algorithm may be used to maximize or
minimize the objective function. Optimization algorithms are
well-known to those skilled in the art and include several commonly
available minimizing or maximizing functions including the Simplex
method and other constrained optimization techniques. It is
understood by those skilled in the art that some minimization
functions are better than others at searching for global minimums,
rather than local minimums. In the optimization process, the
location and size of the cutoff region for each marker may be
allowed to vary to provide at least two degrees of freedom per
marker. Such variable parameters are referred to herein as
independent variables. In a preferred embodiment, the weighting
coefficient for each marker is also allowed to vary across
iterations of the optimization algorithm. In various embodiments,
any permutation of these parameters may be used as independent
variables.
[0099] In addition to the above-described parameters, the sense of
each marker may also be used as an independent variable. For
example, in many cases, it may not be known whether a higher level
for a certain marker is generally indicative of a diseased state or
a non-diseased state. In such a case, it may be useful to allow the
optimization process to search on both sides. In practice, this may
be implemented in several ways. For example, in one embodiment, the
sense may be a truly separate independent variable which may be
flipped between positive and negative by the optimization process.
Alternatively, the sense may be implemented by allowing the
weighting coefficient to be negative.
[0100] The optimization algorithm may be provided with certain
constraints as well. For example, the resulting ROC curve may be
constrained to provide an area-under-curve of greater than a
particular value. ROC curves having an area under the curve of 0.5
indicate complete randomness, while an area under the curve of 1.0
reflects perfect separation of the two sets. Thus, a minimum
acceptable value, such as 0.75, may be used as a constraint,
particularly if the objective function does not incorporate the
area under the curve. Other constraints may include limitations on
the weighting coefficients of particular markers. Additional
constraints may limit the sum of all the weighting coefficients to
a particular value, such as 1.0.
[0101] The iterations of the optimization algorithm generally vary
the independent parameters to satisfy the constraints while
minimizing or maximizing the objective function. The number of
iterations may be limited in the optimization process. Further, the
optimization process may be terminated when the difference in the
objective function between two consecutive iterations is below a
predetermined threshold, thereby indicating that the optimization
algorithm has reached a region of a local minimum or a maximum.
[0102] Thus, the optimization process may provide a panel of
markers including weighting coefficients for each marker and cutoff
regions for the mapping of marker values to indicators. In order to
develop lower-cost panels which require the measurement of fewer
marker levels, certain markers may be eliminated from the panel. In
this regard, the effective contribution of each marker in the panel
may be determined to identify the relative importance of the
markers. In one embodiment, the weighting coefficients resulting
from the optimization process may be used to determine the relative
importance of each marker. The markers with the lowest coefficients
may be eliminated.
[0103] In certain cases, the lower weighting coefficients may not
be indicative of a low importance. Similarly, a higher weighting
coefficient may not be indicative of a high importance. For
example, the optimization process may result in a high coefficient
if the associated marker is irrelevant to the diagnosis. In this
instance, there may not be any advantage that will drive the
coefficient lower. Varying this coefficient may not affect the
value of the objective function.
[0104] Exemplary Marker Panels
[0105] The present invention is described hereinafter generally in
terms of the differential diagnosis of diseases related to dyspnea.
The skilled artisan will understand, however, that the concepts of
symptom-based differential diagnosis described herein are generally
applicable to any physical characteristics that are indicative of a
plurality of possible etiologies such as fever, neurologic
dysfunction, chest pain ("angina"), dizzyness, headache, etc.
[0106] A first step in the identification of suitable markers for
symptom-bases differential diagnosis requires a consideration of
the possible diagnoses that may be causative of the non-specific
symptom observed. In the case of dyspnea, the potential causes are
myriad. In a preferred embodiment, the following discussion
considers three potential diagnoses: congestive heart failure,
pulmonary embolism, and myocardial infarction; and three potential
markers for inclusion in a differential diagnosis panel for these
potential diagnoses: BNP, D-dimer, and cardiac troponin,
respecitively. In another preferred embodiement, markers for three
potential diagnoses, congestive heart failure, pulmonary embolism,
and myocardial infarction include three potential markers in a
differential diagnosis panel, BNP related peptides, D-dimer, and
cardiac troponin, respecitively. In a preferred embodiment, three
potential diagnoses in the case of dyspnea include congestive heart
failure, pulmonary embolism, and myocaridal infarction. In a second
preferred embodiment, four potential diagnoses in the case of
dyspnea include congestive heart failure, pulmonary embolism, and
myocaridal infarction, and atrial fibrillation. Potential markers
for inclusion in a differential diagnosis panel include one or more
of the following: BNP (heart failure), BNP related peptides (heart
failure), D-dimer (pulmonary embolism), cardiac troponin
(myocardial infarction), ANP (atrial fibrillation), and ANP related
peptides (atrial fibrillation).
[0107] BNP
[0108] B-type natriuretic peptide (BNP), also called brain-type
natriuretic peptide is a 32 amino acid, 4 kDa peptide that is
involved in the natriuresis system to regulate blood pressure and
fluid balance. Bonow, R. O., Circulation 93:1946-1950 (1996). The
precursor to BNP is synthesized as a 108-amino acid molecule,
referred to as "pre pro BNP," that is proteolytically processed
into a 76-amino acid N-terminal peptide (amino acids 1-76),
referred to as "NT pro BNP" and the 32-amino acid mature hormone,
referred to as BNP or BNP 32 (amino acids 77-108). It has been
suggested that each of these species--NT pro-BNP, BNP-32, and the
pre pro BNP--can circulate in human plasma. Tateyama et al.,
Biochem. Biophys. Res. Commun. 185: 760-7 (1992); Hunt et al.,
Biochem. Biophys. Res. Commun. 214: 1175-83 (1995). The 2 forms,
pre pro BNP and NT pro BNP, and peptides which are derived from
BNP, pre pro BNP and NT pro BNP and which are present in the blood
as a result of proteolyses of BNP, NT pro BNP and pre pro BNP, are
collectively described as markers related to or associated with
BNP.
[0109] The term "BNP" as used herein refers to the mature 32-amino
acid BNP molecule itself. As the skilled artisan will recognize,
however, because of its relationship to BNP, the concentration of
NT pro-BNP molecule can also provide diagnostic or prognostic
information in patients. The phrases "marker related to BNP" or
"BNP related peptide" refers to any polypeptide that originates
from the pre pro-BNP molecule, other than the 32-amino acid BNP
molecule itself. Proteolytic degradation of BNP and of peptides
related to BNP have also been described in the literature and these
proteolytic fragments are also encompassed it the term "BNP related
peptides."
[0110] BNP and BNP-related peptides are predominantly found in the
secretory granules of the cardiac ventricles, and are released from
the heart in response to both ventricular volume expansion and
pressure overload. Wilkins, M. et al., Lancet 349: 1307-10 (1997).
Elevations of BNP are associated with raised atrial and pulmonary
wedge pressures, reduced ventricular systolic and diastolic
function, left ventricular hypertrophy, and myocardial infarction.
Sagnella, G. A., Clinical Science 95: 519-29 (1998). Furthermore,
there are numerous reports of elevated BNP concentration associated
with congestive heart failure and renal failure. Thus, BNP levels
in a patient may be indicative of several possible underlying
causes of dyspnea.
[0111] D-Dimer
[0112] D-dimer is a crosslinked fibrin degradation product with an
approximate molecular mass of 200 kDa. The normal plasma
concentration of D-dimer is <150 ng/ml (750 pM). The plasma
concentration of D-dimer is elevated in patients with acute
myocardial infarction and unstable angina, but not stable angina.
Hoffmeister, H. M. et al., Circulation 91: 2520-27 (1995);
Bayes-Genis, A. et al., Thromb. Haemost. 81: 865-68 (1999);
Gurfinkel, E. et al., Br. Heart J. 71: 151-55 (1994); Kruskal, J.
B. et al., N. Engl. J. Med. 317: 1361-65 (1987); Tanaka, M. and
Suzuki, A., Thromb. Res. 76: 289-98 (1994).
[0113] The plasma concentration of D-dimer also will be elevated
during any condition associated with coagulation and fibrinolysis
activation, including stroke, surgery, atherosclerosis, trauma, and
thrombotic thrombocytopenic purpura. D-dimer is released into the
bloodstream immediately following proteolytic clot dissolution by
plasmin. The plasma concentration of D-dimer can exceed 2 .mu.g/ml
in patients with unstable angina. Gurfinkel, E. et al., Br. Heart J
71: 151-55 (1994). Plasma D-dimer is a specific marker of
fibrinolysis and indicates the presence of a prothrombotic state
associated with acute myocardial infarction and unstable angina.
The plasma concentration of D-dimer is also nearly always elevated
in patients with acute pulmonary embolism; thus, normal levels of
D-dimer may allow the exclusion of pulmonary embolism. Egermayer et
al., Thorax 53: 830-34 (1998).
[0114] Cardiac Troponin
[0115] Troponin I (TnI) is a 25 kDa inhibitory element of the
troponin complex, found in muscle tissue. TnI binds to actin in the
absence of Ca.sup.2+, inhibiting the ATPase activity of actomyosin.
A TnI isoform that is found in cardiac tissue (cTnI) is 40%
divergent from skeletal muscle TnI, allowing both isoforms to be
immunologically distinguished. The normal plasma concentration of
cTnI is <0.1 ng/ml (4 pM). cTnI is released into the bloodstream
following cardiac cell death; thus, the plasma cTnI concentration
is elevated in patients with acute myocardial infarction.
Investigations into changes in the plasma cTnI concentration in
patients with unstable angina have yielded mixed results, but cTnI
is not elevated in the plasma of individuals with stable angina.
Benamer, H. et al., Am. J. Cardiol. 82: 845-50 (1998); Bertinchant,
J. P. et al., Clin. Biochem. 29: 587-94 (1996); Tanasijevic, M. J.
et al., Clin. Cardiol. 22: 13-16 (1999); Musso, P. et al., J. Ital.
Cardiol. 26: 1013-23 (1996); Holvoet, P. et al., JAMA 281: 1718-21
(1999); Holvoet, P. et al., Circulation 98: 1487-94 (1998).
[0116] The plasma concentration of cTnI in patients with acute
myocardial infarction is significantly elevated 4-6 hours after
onset, peaks between 12-16 hours, and can remain elevated for one
week. The release kinetics of cTnI associated with unstable angina
may be similar. The measurement of specific forms of cardiac
troponin, including free cardiac troponin I and complexes of
cardiac troponin I with troponin C and/or T may provide the user
with the ability to identify various stages of ACS. Free and
complexed cardiac-troponin T may be used in a manner analogous to
that described for cardiac troponin I. Cardiac troponin T complex
may be useful either alone or when expressed as a ratio with total
cardiac troponin I to provide information related to the presence
of progressing myocardial damage. Ongoing ischemia may result in
the release of the cardiac troponin TIC complex, indicating that
higher ratios of cardiac troponin TIC:total cardiac troponin I may
be indicative of continual damage caused by unresolved ischemia.
See, U.S. Pat. Nos. 6,147,688, 6,156,521, 5,947,124, and 5,795,725,
which are hereby incorporated by reference in their entirety,
including all tables, figures, and claims. One skilled in the art
recognizes that in measuring cardiac troponin, one can measure the
different isoforms of troponin I and troponin T.
[0117] One skilled in the art recognizes that in measuring cardiac
troponin, one can measure the different isoforms of troponin I and
troponin T. Thus, one may preferably measure free cardiac troponin
I, free cardiac troponin T, cardiac troponin I in a complex
comprising one or both of troponin T and troponin C, cardiac
troponin T in a complex comprising one or both of troponin I and
troponin C, total cardiac troponin I (meaning free and complexed
cardiac troponin I), and/or total cardiac troponin T, The term "at
least one cardiac troponin form" as used herein refers to any one
of these foregoing forms.
[0118] ANP
[0119] A-type natriuretic peptide (ANP) (also referred to as atrial
natriuretic peptide or cardiodilatin Forssmann et al Histochem Cell
Biol 110: 335-357 (1998)) is a 28 amino acid peptide that is
synthesized, stored, and released atrial myocytes in response to
atrial distension, angiotensin II stimulation, endothelin, and
sympathetic stimulation (beta-adrenoceptor mediated). ANP is
synthesized as a precursor molecule (pro-ANP) that is converted to
an active form, ANP, by proteolytic cleavage and also forming
N-terminal ANP (1-98). N-terminal ANP and ANP have been reported to
increase in patients exhibiting atrial fibrillation and heart
failure (Rossi et al. Journal of the American College of Cardiology
35: 1256-62 (2000). In addition to atrial natriuretic peptide
(ANP99-126) itself, linear peptide fragments from its N-terminal
prohormone segment have also been reported to have biological
activity. As the skilled artisan will recognize, however, because
of its relationship to ANP, the concentration of N-terminal ANP
molecule can also provide diagnostic or prognostic information in
patients. The phrase "marker related to ANP or ANP related peptide"
refers to any polypeptide that originates from the pro-ANP molecule
(1-126), other than the 28-amino acid ANP molecule itself.
Proteolytic degradation of ANP and of peptides related to ANP have
also been described in the literature and these proteolytic
fragments are also encompassed it the term "ANP related
peptides."
[0120] Elevated levels of ANP are found during hypervolemia, atrial
fibrillation and congestive heart failure. ANP is involved in the
long-term regulation of sodium and water balance, blood volume and
arterial pressure. This hormone decreases aldosterone release by
the adrenal cortex, increases glomerular filtration rate (GFR),
produces natriuresis and diuresis (potassium sparing), and
decreases renin release thereby decreasing angiotensin II. These
actions contribute to reductions in blood volume and therefore
central venous pressure (CVP), cardiac output, and arterial blood
pressure. Several isoforms of ANP have been identified, and their
relationship to stroke incidence studied. See, e.g., Rubatu et al.,
Circulation 100:1722-6, 1999; Estrada et al., Am. J. Hypertens.
7:1085-9, 1994.
[0121] Chronic elevations of ANP appear to decrease arterial blood
pressure primarily by decreasing systemic vascular resistance. The
mechanism of systemic vasodilation may involve ANP
receptor-mediated elevations in vascular smooth muscle cGMP as well
as by attenuating sympathetic vascular tone. This latter mechanism
may involve ANP acting upon sites within the central nervous system
as well as through inhibition of norepinephrine release by
sympathetic nerve terminals. ANP may be viewed as a
counter-regulatory system for the renin-angiotensin system. A new
class of drugs that are neutral endopeptidase (NEP) inhibitors have
demonstrated efficacy in heart failure. These drugs inhibit neutral
endopeptidase, the enzyme responsible for the degradation of ANP,
and thereby elevate plasma levels of ANP. NEP inhibition is
particularly effective in heart failure when the drug has a
combination of both NEP and ACE inhibitor properties.
[0122] Based on the foregoing discussion, the skilled artisan will
recognize that, for example, increased BNP is indicative of
congestive heart failure, but may also be indicative of other
cardiac-related conditions such as myocardial infarction. Thus, the
inclusion of a marker related to myocardial injury such as cardiac
troponin I and/or cardiac troponin T can permit further
discrimination of the disease underlying the observed dyspnea and
the increased BNP level. In this case, an increased level of
cardiac troponin may be used to rule in myocardial infarction.
[0123] Similarly, BNP may also be indicative of pulmonary embolism.
The inclusion of a marker related to coagulation and hemostasis
such as D-dimer can permit further discrimination of the disease
underlying the observed dyspnea and the increased BNP level. In
this case, a normal level of D-dimer may be used to rule out
pulmonary embolism.
[0124] A detailed analysis of this exemplary marker panel is
provided in the following examples. The skilled artisan will
readily acknowledge that other markers may be substituted in or
added to this marker panel to further discriminate the causes of
dyspnea in accordance with the methods for identification and use
of diagnostic markers described herein. Additional suitable markers
are described in the following sections.
[0125] As discussed in detail herein, the foregoing principles of
marker panel design may be applied broadly to symptom-based
differential diagnosis. For example, in the case of abdominal pain,
the plurality of markers are preferably selected to rule in or out
a plurality of the following: aortic dissection, mesenteric
embolism, pancreatitis, appendicitis, angina, myocardial
infarction, one or more infectious diseases described above,
influenza, esophageal carcinoma, gastric adenocarcinoma, colorectal
adenocarcinoma, pancreatic tumors including ductal adenocarcinoma,
cystadenocarcinoma, and insulinoma. In a preferred embodiment, the
potential diagnoses for abdominal pain include aortic aneurysm,
mesenteric embolism, pancreatitis, appendicitis, angina and
myocardial infarction.
[0126] The foregoing principles may also be applied to subdivide
differential diagnosis to a given level of detail required by the
clinical artisan. For example, the differential diagnosis of
various symptoms may require discrimination between heart failure
and atrial fibrillation. An exemplary marker panel for performing
such discrimination preferably includes BNP or BNP related
peptides, and ANP or ANP related peptides, respectively. Additional
markers may be defined to distinguish between systolic and
diastolic dysfunction and atrial fibrillation. Preferred markers in
this case include BNP, calcitonin gene related peptide, calcitonin
and urotensin 1 for differentiation of systolic and diastolic
dysfunction and ANP or ANP related peptides for the detection of
atrial fibrillation. Likewise, markers may be defined to
distinguish between systolic and diastolic dysfunction, atrial
fibrillation, myocardial ischemia and cardiac necrosis. Preferred
markers in this case include BNP, calcitonin gene related peptide,
calcitonin and urotensin 1 for differentiation of systolic and
diastolic dysfunction and ANP or ANP related peptides for the
detection of atrial fibrillation and BNP and cardiac troponins for
the detection of myocardial ischemia and necrosis.
[0127] In the case of chest pain, the present invention can provide
markers able to distinguish between aortic dissection, myocardial
ischemia, and cardiac necrosis; markers able to distinguish between
aortic dissection, myocardial ischemia, and myocardial infarction;
markers able to distinguish between aortic dissection, myocardial
ischemia, cardiac necrosis and heart failure; markers able to
distinguish between aortic dissection, myocardial ischemia, cardiac
necrosis and myocardial infarction; markers able to distinguish
between aortic dissection, myocardial ischemia, cardiac necrosis
and atrial fibrillation; and/or markers able to distinguish between
aortic dissection, myocardial ischemia and cardiac necrosis,
myocardial infarction and atrial fibrillation. In accordance with
the foregoing, a particularly preferred marker for aortic
dissection is smooth muscle myosin, and most preferably smooth
muscle myosin heavy chain, and a particularly preferred marker for
atrial fibrillation is ANP or an ANP-related marker.
[0128] Preferred marker sets are those comprising smooth muscle
myosin heavy chain and ANP or an ANP-related marker to distinguish
aortic dissection and atrial fibrillation, respectively; smooth
muscle myosin heavy chain, ANP or an ANP-related marker, and BNP or
a BNP-related marker to distinguish aortic dissection, atrial
fibrillation and myocardial ischemia, respectively; smooth muscle
myosin heavy chain, BNP or a BNP-related marker, and a cardiac
troponin form to distinguish aortic dissection, myocardial
ischemia, and myocardial infarction, respectively; and smooth
muscle myosin heavy chain, BNP or a BNP-related marker, creatine
kinase MB, myoglobin, and a cardiac troponin form to distinguish
aortic dissection, myocardial ischemia, cardiac necrosis, and
myocardial infarction.
[0129] Similarly, in the case of disturbanes of metabolic state,
the plurality of markers are preferably selected to rule in or out
a plurality of the following: diabetes mellitus, diabetic
ketoacidosis, alcoholic ketoacidosis, respiratory acidosis,
respiratory alkalosis, nonketogenic hyperglycemia, hypoglycemia,
renal failure, interstitial renal disease, COPD, pneumonia,
pulmonary edema and asthma.
[0130] In the case of neurologic dysfunction, the plurality of
markers are preferably selected to rule in or out a plurality of
the following: stroke, brain tumor, cerebral hypoxia, hypoglycemia,
migraine, atrial fibrillation, myocardial infarction, cardiac
ischemia, peripheral vascular disease and seizure. Preferred
markers in this case include specific markers of cerebral injury
such as adenylate kinase, brain-derived neurotrophic factor,
calbindin-D, creatine kinase-BB, glial fibrillary acidic protein,
lactate dehydrogenase, myelin basic protein, neural cell adhesion
molecule, neuron-specific enolase, neurotrophin-3, proteolipid
protein, S-100b, thrombomodulin, protein kinase C gamma; and/or one
or more non-specific markers of cerebral injury such as
.beta.-thromboglobulin, D-dimer, fibrinopeptide A,
plasmin-.alpha.-2-antiplasmin complex, platelet factor 4,
prothrombin fragment 1+2, thrombin-antithrombin III complex, tissue
factor, von Willebrand factor, adrenomedullin, cardiac troponin I
(for myocardial ischemia and necrosis), head activator, hemoglobin
.alpha..sub.2 chain, caspase-3, vascular endothelial growth factor
(VEGF), one or more endothelins (e.g., endothelin-1, endothelin-2,
and endothelin-3), interleukin-8, Atrial natriuretic peptide,
B-type natriuretic peptide (for myocardial ischemia and necrosis),
and C-type natriuretic peptide; and/or one or more acute phase
reactants such as C-reactive protein, ceruloplasmin, fibrinogen,
.alpha.1-acid glycoprotein, .alpha..sub.2-antitrypsin, haptoglobin,
insulin-like growth factor-1, interleukin-1.beta., interleukin-1
receptor antagonist, interleukin-6, transforming growth factor,
tumor necrosis factor .alpha., E-selectin, intercellular adhesion
molecule-1, matrix metalloproteinases (e.g., matrix
metalloproteinase 9 (MMP-9)), monocyte chemotactic protein-1, and
vascular cell adhesion molecule.
[0131] Methods and marker sets for differential diagnosis of stroke
and other cerebral injuries are described in U.S. patent Ser. No.
10/225,082, filed Aug. 20, 2002, which is hereby incorporated in
its entirety, including all tables figures and claims. As described
therein, preferred marker panels diagnose and/or differentiate
between stroke, subarachnoid hemorrhage, intracerebral hemorrhage,
and/or hemorrhagic stroke; and/or can distinguish between ischemic
and hemorrhagic stroke. Particularly preferred are markers that
differentiate between thrombotic, embolic, lacunar, hypoperfusion,
intracerebral hemorrhage, and subarachnoid hemorrhage types of
strokes. Particularly preferred marker sets include BNP, IL-6,
S-100b, MMP-9, TAT complex, and vWF A1-integrin; BNP, S-100b,
MMP-9, and vWF-A1-integrin; vWF-A1, VEGF, and MMP-9; caspase-3,
MMP-9, and GFAP; caspase-3, MMP-9, vWF-A1, and BNP; NCAM, BDNF,
Caspase-3, MMP-9, vWF-A1, and VEGF; NCAM, BDNF, Caspase-3, MMP-9,
vWF-A1, and S-100b; VEGF; NCAM, BDNF, Caspase-3, MMP-9, vWF-A1, and
MCP-1; VEGF; NCAM, BDNF, Caspase-3, MMP-9, VEGF, and vWF
A1-integrin; BDNF, MMP-9, S-100b, vWF A1-integrin, MCP-1, and GFAP;
BDNF, caspase-3, MMP-9, vWF-A1, S-100b, and GFAP; NCAM, BDNF,
MMP-9, vWF-A1, S-100b, and GFAP; NCAM, BDNF, caspase-3, MMP-9,
S-100P, and GFAP; caspase-3, NCAM, MCP-1, S100b, MMP-9, vWF
A1-integrin, and BNP; caspase-3, NCAM, MCP-1, S100b, MMP-9, vWF A1,
BNP, and GFAP; CRP, NT-3, vWF, MMP-9, VEGF, and CKBB; CRP, MMP-9,
VEGF, CKBB, and MCP-1; CRP, NT-3, MMP-9, VEGF, CKBB, and MCP-1; and
CRP, MMP-9, VEGF, CKBB, MCP-1. Calbindin, vWF VP1, vWF A3, vWF
A1-A3, TAT complex, proteolipid protein, IL-6, IL-8, myelin basic
protein, S-100b, tissue factor, GFAP, vWF A1-integrin, CNP, and
NCAM.
[0132] A panel consisting of the markers referenced herein may be
constructed to provide relevant information related to the
differential diagnosis of interest. Such a panel may be constructed
using 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19 or 20 individual markers. The analysis of a single marker or
subsets of markers comprising a larger panel of markers could be
carried out by one skilled in the art to optimize clinical
sensitivity or specificity in various clinical settings. These
include, but are not limited to ambulatory, urgent care, critical
care, intensive care, monitoring unit, inpatient, outpatient,
physician office, medical clinic, and health screening settings.
Furthermore, one skilled in the art can use a single marker or a
subset of markers comprising a larger panel of markers in
combination with an adjustment of the diagnostic threshold in each
of the aforementioned settings to optimize clinical sensitivity and
specificity. The clinical sensitivity of an assay is defined as the
percentage of those with the disease that the assay correctly
predicts, and the specificity of an assay is defined as the
percentage of those without the disease that the assay corrects
predicts (Tietz Textbook of Clinical Chemistry, 2.sup.nd edition,
Carl Burtis and Edward Ashwood eds., W.B. Saunders and Company, p.
496). The following provides a brief discussion of additional
exemplary markers for use in identifying suitable marker panels by
the methods described herein.
[0133] (i) Exemplary Markers Related to Myocardial Injury
[0134] Annexin V, also called lipocortin V, endonexin II,
calphobindin I, calcium binding protein 33, placental anticoagulant
protein I, thromboplastin inhibitor, vascular
anticoagulant-.alpha., and anchorin CII, is a 33 kDa
calcium-binding protein that is an indirect inhibitor and regulator
of tissue factor. Annexin V is composed of four homologous repeats
with a consensus sequence common to all annexin family members,
binds calcium and phosphatidyl serine, and is expressed in a wide
variety of tissues, including heart, skeletal muscle, liver, and
endothelial cells (Giambanco, I. et al., J. Histochem. Cytochem.
39:P1189-1198, 1991; Doubell, A. F. et al., Cardiovasc. Res.
27:1359-1367, 1993). The normal plasma concentration of annexin V
is <2 ng/ml (Kaneko, N. et al., Clin. Chim. Acta 251:65-80,
1996). The plasma concentration of annexin V is elevated in
individuals with acute myocardial infarction (Kaneko, N. et al.,
Clin. Chim. Acta 251:65-80, 1996). Due to its wide tissue
distribution, elevation of the plasma concentration of annexin V
may be associated with any condition involving non-cardiac tissue
injury. However, one study has found that plasma annexin V
concentrations were not significantly elevated in patients with old
myocardial infarction, chest pain syndrome, valvular heart disease,
lung disease, and kidney disease (Kaneko, N. et al., Clin. Chim.
Acta 251:65-80, 1996). Annexin V is released into the bloodstream
soon after acute myocardial infarction onset. The annexin V
concentration in the plasma of acute myocardial infarction patients
decreased from initial (admission) values, suggesting that it is
rapidly cleared from the bloodstream (Kaneko, N. et al. Clin. Chim.
Acta 251:65-80, 1996).
[0135] Enolase is a 78 kDa homo- or heterodimeric cytosolic protein
produced from .alpha., .beta., and .gamma. subunits. Enolase
catalyzes the interconversion of 2-phosphoglycerate and
phosphoenolpyruvate in the glycolytic pathway. Enolase is present
as .alpha..alpha., .alpha..beta., .beta..beta., .alpha..gamma. and
.gamma..gamma. isoforms. The .alpha. subunit is found in most
tissues, the .beta. subunit is found in cardiac and skeletal
muscle, and the .gamma. subunit is found primarily in neuronal and
neuroendocrine tissues. .beta.-enolase is composed of .alpha..beta.
and .beta..beta. enolase, and is specific for muscle. The normal
plasma concentration of .beta.-enolase is <10 ng/ml (120 pM).
.beta.-enolase is elevated in the serum of individuals with acute
myocardial infarction, but not in individuals with angina (Nomura,
M. et al., Br. Heart J. 58:29-33, 1987; Herraez-Dominguez, M. V. et
al., Clin. Chim. Acta 64:307-315, 1975). Further investigations
into possible changes in plasma .beta.-enolase concentration
associated with unstable and stable angina need to be performed.
The plasma concentration of .beta.-enolase is elevated during heart
surgery, muscular dystrophy, and skeletal muscle injury (Usui, A.
et al., Cardiovasc. Res. 23:737-740, 1989; Kato, K. et al., Clin.
Chim. Acta 131:75-85, 1983; Matsuda, H. et al., Forensic Sci. Int.
99:197-208, 1999). .beta.-enolase is released into the bloodstream
immediately following cardiac or skeletal muscle injury. The plasma
.beta.-enolase concentration was elevated to more than 150 ng/ml in
the perioperative stage of cardiac surgery, and remained elevated
for 1 week. Serum .beta.-enolase concentrations peaked
approximately 12-14 hours after the onset of chest pain and acute
myocardial infarction and approached baseline after 1 week had
elapsed from onset, with maximum levels approaching 1 .mu.g/ml
(Kato, K. et al., Clin. Chim. Acta 131:75-85, 1983; Nomura, M. et
al., Br. Heart J. 58:29-33, 1987).
[0136] Creatine kinase (CK) is a 85 kDa cytosolic enzyme that
catalyzes the reversible formation ADP and phosphocreatine from ATP
and creatine. CK is a homo- or heterodimer composed of M and B
chains. CK-MB is the isoform that is most specific for cardiac
tissue, but it is also present in skeletal muscle and other
tissues. The normal plasma concentration of CK-MB is <5 ng/ml.
The plasma CK-MB concentration is significantly elevated in
patients with acute myocardial infarction. Plasma CK-MB is not
elevated in patients with stable angina, and investigation into
plasma CK-MB concentration elevations in patients with unstable
angina have yielded mixed results (Thygesen, K. et al., Eur. J.
Clin. Invest. 16:1-4, 1986; Koukkunen, H. et al., Ann. Med.
30:488-496, 1998; Bertinchant, J. P. et al., Clin. Biochem.
29:587-594, 1996; Benamer, H. et al., Am. J. Cardiol. 82:845-850,
1998; Norregaard-Hansen, K. et al., Eur. Heart J. 13:188-193,
1992). The mixed results associated with unstable angina suggest
that CK-MB may be useful in determining the severity of unstable
angina because the extent of myocardial ischemia is directly
proportional to unstable angina severity. Elevations of the plasma
CK-MB concentration are associated with skeletal muscle injury and
renal disease. CK-MB is released into the bloodstream following
cardiac cell death. The plasma concentration of CK-MB in patients
with acute myocardial infarction is significantly elevated 4-6
hours after onset, peaks between 12-24 hours, and returns to
baseline after 3 days. The release kinetics of CK-MB associated
with unstable angina may be similar.
[0137] Glycogen phosphorylase (GP) is a 188 kDa intracellular
allosteric enzyme that catalyzes the removal of glucose (liberated
as glucose-1-phosphate) from the nonreducing ends of glycogen in
the presence of inorganic phosphate during glycogenolysis. GP is
present as a homodimer, which associates with another homodimer to
form a tetrameric enzymatically active phosphorylase A. There are
three isoforms of GP that can be immunologically distinguished. The
BB isoform is found in brain and cardiac tissue, the MM isoform is
found in skeletal muscle and cardiac tissue, and the LL isoform is
predominantly found in liver (Mair, J. et al., Br. Heart J.
72:125-127, 1994). GP-BB is normally associated with the
sarcoplasmic reticulum glycogenolysis complex, and this association
is dependent upon the metabolic state of the myocardium (Mair, J.,
Clin. Chim. Acta 272:79-86, 1998). At the onset of hypoxia,
glycogen is broken down, and GP-BB is converted from a bound form
to a free cytoplasmic form (Krause, E. G. et al. Mol. Cell Biochem.
160-161:289-295, 1996). The normal plasma GP-BB concentration is
<7 ng/ml (36 pM). The plasma GP-BB concentration is
significantly elevated in patients with acute myocardial infarction
and unstable angina with transient ST-T elevations, but not stable
angina (Mair, J. et al., Br. Heart J. 72:125-127, 1994; Mair, J.,
Clin. Chim. Acta 272:79-86, 1998; Rabitzsch, G. et al., Clin. Chem.
41:966-978, 1995; Rabitzsch, G. et al., Lancet 341:1032-1033,
1993). Furthermore, GP-BB also can be used to detect perioperative
acute myocardial infarction and myocardial ischemia in patients
undergoing coronary artery bypass surgery (Rabitzsch, G. et al.,
Biomed. Biochim. Acta 46:S584-S588, 1987; Mair, P. et al., Eur. J.
Clin. Chem. Clin. Biochem. 32:543-547, 1994). GP-BB has been
demonstrated to be a more sensitive marker of unstable angina and
acute myocardial infarction early after onset than CK-MB, cardiac
tropopnin T, and myoglobin (Rabitzsch, G. et al., Clin. Chem.
41:966-978, 1995). Because it is also found in the brain, the
plasma GP-BB concentration also may be elevated during ischemic
cerebral injury. GP-BB is released into the bloodstream under
ischemic conditions that also involve an increase in the
permeability of the cell membrane, usually a result of cellular
necrosis. GP-BB is significantly elevated within 4 hours of chest
pain onset in individuals with unstable angina and transient ST-T
ECG alterations, and is significantly elevated while myoglobin,
CK-MB, and cardiac troponin T are still within normal levels (Mair,
J. et al., Br. Heart J 72:125-127, 1994). Furthermore, GP-BB can be
significantly elevated 1-2 hours after chest pain onset in patients
with acute myocardial infarction (Rabitzsch, G. et al., Lancet
341:1032-1033, 1993). The plasma GP-BB concentration in patients
with unstable angina and acute myocardial infarction can exceed 50
ng/ml (250 pM) (Mair, J. et al., Br. Heart J 72:125-127, 1994;
Mair, J., Clin. Chim. Acta 272:79-86, 1998; Krause, E. G. et al.,
Mol. Cell Biochem. 160-161:289-295, 1996; Rabitzsch, G. et al.,
Clin. Chem. 41:966-978, 1995; Rabitzsch, G. et al., Lancet
341:1032-1033, 1993). GP-BB appears to be a very sensitive marker
of myocardial ischemia, with specificity similar to that of CK-BB.
GP-BB plasma concentrations are elevated within the first 4 hours
after acute myocardial infarction onset, which suggests that it may
be a very useful early marker of myocardial damage. Furthermore,
GP-BB is not only a more specific marker of cardiac tissue damage,
but also ischemia, since it is released to an unbound form during
myocardial ischemia and would not normally be released upon
traumatic injury. This is best illustrated by the usefulness of
GP-BB in detecting myocardial ischemia during cardiac surgery.
GP-BB may be a very useful marker of early myocardial ischemia
during acute myocardial infarction and severe unstable angina.
[0138] Heart-type fatty acid binding protein (H-FABP) is a
cytosolic 15 kDa lipid-binding protein involved in lipid
metabolism. Heart-type FABP antigen is found not only in heart
tissue, but also in kidney, skeletal muscle, aorta, adrenals,
placenta, and brain (Veerkamp, J. H. and Maatman, R. G., Prog.
Lipid Res. 34:17-52, 1995; Yoshimoto, K. et al., Heart Vessels
10:304-309, 1995). Furthermore, heart-type FABP mRNA can be found
in testes, ovary, lung, mammary gland, and stomach (Veerkamp, J. H.
and Maatman, R. G., Prog. Lipid Res. 34:17-52, 1995). The normal
plasma concentration of FABP is <6 ng/ml (400 pM). The plasma
H-FABP concentration is elevated in patients with acute myocardial
infarction and unstable angina (Ishii, J. et al., Clin. Chem.
43:1372-1378, 1997; Tsuji, R. et al., Int. J. Cardiol. 41:209-217,
1993). Furthermore, H-FABP may be useful in estimating infarct size
in patients with acute myocardial infarction (Glatz, J. F. et al.,
Br. Heart J. 71:135-140, 1994). Myocardial tissue as a source of
H-FABP can be confirmed by determining the ratio of myoglobin/FABP
(grams/grams). A ratio of approximately 5 indicates that FABP is of
myocardial origin, while a higher ratio indicates skeletal muscle
sources (Van Nieuwenhoven, F. A. et al., Circulation 92:2848-2854,
1995). Because of the presence of H-FABP in skeletal muscle, kidney
and brain, elevations in the plasma H-FABP concentration may be
associated with skeletal muscle injury, renal disease, or stroke.
H-FABP is released into the bloodstream following cardiac tissue
necrosis. The plasma H-FABP concentration can be significantly
elevated 1-2 hours after the onset of chest pain, earlier than
CK-MB and myoglobin (Tsuji, R. et al., Int. J. Cardiol. 41:209-217,
1993; Van Nieuwenhoven, F. A. et al., Circulation 92:2848-2854,
1995; Tanaka, T. et al., Clin. Biochem. 24:195-201, 1991).
Additionally, H-FABP is rapidly cleared from the bloodstream, and
plasma concentrations return to baseline after 24 hours after acute
myocardial infarction onset (Glatz, J. F. et al., Br. Heart J.
71:135-140, 1994; Tanaka, T. et al., Clin. Biochem. 24:195-201,
1991).
[0139] Phosphoglyceric acid mutase (PGAM) is a 57 kDa homo- or
heterodimeric intracellular glycolytic enzyme composed of 29 kDa M
or B subunits that catalyzes the interconversion of
3-phosphoglycerate to 2-phosphoglycerate in the presence of
magnesium. Cardiac tissue contains isozymes MM, MB, and BB,
skeletal muscle contains primarily PGAM-MM, and most other tissues
contain PGAM-BB (Durany, N. and Carreras, J., Comp. Biochem.
Physiol. B. Biochem. Mol. Biol. 114:217-223, 1996). Thus, PGAM-MB
is the most specific isozyme for cardiac tissue. PGAM is elevated
in the plasma of patients with acute myocardial infarction, but
further studies need to be performed to determine changes in the
plasma PGAM concentration associated with acute myocardial
infarction, unstable angina and stable angina (Mair, J., Crit. Rev.
Clin. Lab. Sci. 34:1-66, 1997). Plasma PGAM-MB concentration
elevations may be associated with unrelated myocardial or possibly
skeletal tissue damage. PGAM-MB is most likely released into the
circulation following cellular necrosis. PGAM has a half-life of
less than 2 hours in the bloodstream of rats (Grisolia, S. et al.,
Physiol. Chem. Phys. 8:37-52, 1976).
[0140] S-100 is a 21 kDa homo- or heterodimeric cytosolic
Ca.sup.2+-binding protein produced from .alpha. and .beta.
subunits. It is thought to participate in the activation of
cellular processes along the Ca.sup.2+-dependent signal
transduction pathway (Bonfrer, J. M. et al., Br. J. Cancer
77:2210-2214, 1998). S-100ao (.alpha..alpha. isoform) is found in
striated muscles, heart and kidney, S-100a (.alpha..beta. isoform)
is found in glial cells, but not in Schwann cells, and S-100b
(.beta..beta. isoform) is found in high concentrations in glial
cells and Schwann cells, where it is a major cytosolic component
(Kato, K. and Kimura, S., Biochim. Biophys. Acta 842:146-150, 1985;
Hasegawa, S. et al., Eur. Urol. 24:393-396, 1993). The normal serum
concentration of S-100ao is <0.25 ng/ml (12 pM), and its
concentration may be influenced by age and sex, with higher
concentrations in males and older individuals (Kikuchi, T. et al.,
Hinyokika Kiyo 36:1117-1123, 1990; Morita, T. et al., Nippon
Hinyokika Gakkai Zasshi 81:1162-1167, 1990; Usui, A. et al., Clin.
Chem. 36:639-641, 1990). The serum concentration of S-100ao is
elevated in patients with acute myocardial infarction, but not in
patients with angina pectoris with suspected acute myocardial
infarction (Usui, A. et al., Clin. Chem. 36:639-641, 1990). Further
investigation is needed to determine changes in the plasma
concentration of S-100ao associated with unstable and stable
angina. Serum S-100ao is elevated in the serum of patients with
renal cell carcinoma, bladder tumor, renal failure, and prostate
cancer, as well as in patients undergoing open heart surgery
(Hasegawa, S. et al., Eur. Urol. 24:393-396, 1993; Kikuchi, T. et
al., Hinyokika Kiyo 36:1117-1123, 1990; Morita, T. et al., Nippon
Hinyokika Gakkai Zasshi 81:1162-1167, 1990; Usui, A. et al., Clin.
Chem. 35:1942-1944, 1989). S-100ao is a cytosolic protein that will
be released into the extracellular space following cell death. The
serum concentration of S-100ao is significantly elevated on
admission in patients with acute myocardial infarction, increases
to peak levels 8 hours after admission, decreases and returns to
baseline one week later (Usui, A. et al., Clin. Chem. 36:639-641,
1990). Furthermore, S-100ao appears to be significantly elevated
earlier after acute myocardial infarction onset than CK-MB (Usui,
A. et al., Clin. Chem. 36:639-641, 1990). The maximum serum S-100ao
concentration can exceed 100 ng/ml. S-100ao may be rapidly cleared
from the bloodstream by the kidney, as suggested by the rapid
decrease of the serum S-100ao concentration of heart surgery
patients following reperfusion and its increased urine
concentration. S-100ao is found in high concentration in cardiac
tissue and appears to be a sensitive marker of cardiac injury.
Major sources of non-specificity of this marker include skeletal
muscle and renal tissue injury. S-100ao may be significantly
elevated soon after acute myocardial infarction onset, and it may
allow for the discrimination of acute myocardial infarction from
unstable angina. Patients with angina pectoris and suspected acute
myocardial infarction, indicating that they were suffering chest
pain associated with an ischemic episode, did not have a
significantly elevated S-100ao concentration.
[0141] Myoglobin is a small (.about.17.8 kDa) heme-containing
protein responsible for the oxygen deposition in muscle tissue. The
small molecular weight of myoglobin allows it to be rapidly
released from muscle tissue when the tissue is damaged. By
comparison, creatine kinase and its isoform CK-MB are larger than
myoglobin and are released more slowly after an acute myocardial
infarction. Because myoglobin escapes rapidly from damaged
myocardial cells, it can be detected as soon as 1 hour after an
acute myocardial infarction, with peak serum levels occurring in 3
to 15 hours The same form of myoglobin is expressed in cardiac and
in skeletal muscle tissues. Because of its lower cardiac
specificity, it is often used together with cardiac Troponin I or
Troponin T. Although using early detection of elevated myoglobin
levels in patients with chest pain to determine whether an AMI has
occurred sounds ideal, some disadvantages are apparent. Because
both cardiac and skeletal muscle contain myoglobin, many
non-cardiac-related factors, such as skeletal muscle or
neuromuscular disorders, strenuous exercise, renal failure,
intramuscular injections, and cardiac bypass surgery, can elevate
serum levels of the protein. Controversy also exists about the
level at which myoglobin becomes indicative of an acute myocardial
infarction, with the reported reference ranges varying from 50 to
120 .mu.g/mL. See, e.g., Montague and Kircher, Am J Clin Pathol.
104:472-476 (1995); Bhayana and Henderson, Clin Biochem. 28:1-29
(1995); Wong, Ann Clin Lab Sci. 26:301-312 (1996); and Selker et
al., Ann Emerg Med. 29:63-69 (1997).
[0142] (ii) Exemplary Markers Related to Coagulation and
Hemostasis
[0143] Plasmin is a 78 kDa serine proteinase that proteolytically
digests crosslinked fibrin, resulting in clot dissolution. The 70
kDa serine proteinase inhibitor .alpha.2-antiplasmin .alpha.2AP)
regulates plasmin activity by forming a covalent 1:1 stoichiometric
complex with plasmin. The resulting .about.150 kDa
plasmin-.alpha.2AP complex (PAP), also called plasmin inhibitory
complex (PIC) is formed immediately after .alpha.2AP comes in
contact with plasmin that is activated during fibrinolysis. The
normal serum concentration of PAP is <1 .mu.g/ml (6.9 nM).
Elevations in the serum concentration of PAP can be attributed to
the activation of fibrinolysis. Elevations in the serum
concentration of PAP may be associated with clot presence, or any
condition that causes or is a result of fibrinolysis activation.
These conditions can include atherosclerosis, disseminated
intravascular coagulation, acute myocardial infarction, surgery,
trauma, unstable angina, stroke, and thrombotic thrombocytopenic
purpura. PAP is formed immediately following proteolytic activation
of plasmin. PAP is a specific marker for fibrinolysis activation
and the presence of a recent or continual hypercoagulable
state.
[0144] .beta.-thromboglobulin (.beta.TG) is a 36 kDa platelet
.alpha. granule component that is released upon platelet
activation. The normal plasma concentration of .beta.TG is <40
ng/ml (1.1 nM). Plasma levels of .beta.-TG appear to be elevated in
patients with unstable angina and acute myocardial infarction, but
not stable angina (De Caterina, R. et al., Eur. Heart J. 9:913-922,
1988; Bazzan, M. et al., Cardiologia 34, 217-220, 1989). Plasma
.beta.-TG elevations also seem to be correlated with episodes of
ischemia in patients with unstable angina (Sobel, M. et al.,
Circulation 63:300-306, 1981). Elevations in the plasma
concentration of .beta.TG may be associated with clot presence, or
any condition that causes platelet activation. These conditions can
include atherosclerosis, disseminated intravascular coagulation,
surgery, trauma, and thrombotic thrombocytopenic purpura, and
stroke (Landi, G. et al., Neurology 37:1667-1671, 1987). .beta.TG
is released into the circulation immediately after platelet
activation and aggregation. It has a biphasic half-life of 10
minutes, followed by an extended 1 hour half-life in plasma
(Switalska, H. I. et al., J. Lab. Clin. Med. 106:690-700, 1985).
Plasma .beta.TG concentration is reportedly elevated dring unstable
angina and acute myocardial infarction. Special precautions must be
taken to avoid platelet activation during the blood sampling
process. Platelet activation is common during regular blood
sampling, and could lead to artificial elevations of plasma
.beta.TG concentration. In addition, the amount of .beta.TG
released into the bloodstream is dependent on the platelet count of
the individual, which can be quite variable. Plasma concentrations
of .beta.TG associated with ACS can approach 70 ng/ml (2 nM), but
this value may be influenced by platelet activation during the
sampling procedure.
[0145] Platelet factor 4 (PF4) is a 40 kDa platelet .alpha. granule
component that is released upon platelet activation. PF4 is a
marker of platelet activation and has the ability to bind and
neutralize heparin. The normal plasma concentration of PF4 is <7
ng/ml (175 pM). The plasma concentration of PF4 appears to be
elevated in patients with acute myocardial infarction and unstable
angina, but not stable angina (Gallino, A. et al., Am. Heart J.
112:285-290, 1986; Sakata, K. et al., Jpn. Circ. J. 60:277-284,
1996; Bazzan, M. et al., Cardiologia 34:217-220, 1989). Plasma PF4
elevations also seem to be correlated with episodes of ischemia in
patients with unstable angina (Sobel, M. et al., Circulation
63:300-306, 1981). Elevations in the plasma concentration of PF4
may be associated with clot presence, or any condition that causes
platelet activation. These conditions can include atherosclerosis,
disseminated intravascular coagulation, surgery, trauma, thrombotic
thrombocytopenic purpura, and acute stroke (Carter, A. M. et al.,
Arterioscler. Thromb. Vasc. Biol. 18:1124-1131, 1998). PF4 is
released into the circulation immediately after platelet activation
and aggregation. It has a biphasic half-life of 1 minute, followed
by an extended 20 minute half-life in plasma. The half-life of PF4
in plasma can be extended to 20-40 minutes by the presence of
heparin (Rucinski, B. et al., Am. J. Physiol. 251:H800-H807, 1986).
Plasma PF4 concentration is reportedly elevated during unstable
angina and acute myocardial infarction, but these studies may not
be completely reliable. Special precautions must be taken to avoid
platelet activation during the blood sampling process. Platelet
activation is common during regular blood sampling, and could lead
to artificial elevations of plasma PF4 concentration. In addition,
the amount of PF4 released into the bloodstream is dependent on the
platelet count of the individual, which can be quite variable.
Plasma concentrations of PF4 associated with disease can exceed 100
ng/ml (2.5 nM), but it is likely that this value may be influenced
by platelet activation during the sampling procedure.
[0146] Fibrinopeptide A (FPA) is a 16 amino acid, 1.5 kDa peptide
that is liberated from amino terminus of fibrinogen by the action
of thrombin. Fibrinogen is synthesized and secreted by the liver.
The normal plasma concentration of FPA is <5 ng/ml (3.3 nM). The
plasma FPA concentration is elevated in patients with acute
myocardial infarction, unstable angina, and variant angina, but not
stable angina (Gensini, G. F. et al., Thromb. Res. 50:517-525,
1988; Gallino, A. et al., Am. Heart J 112:285-290, 1986; Sakata, K.
et al., Jpn. Circ. J. 60:277-284, 1996; Theroux, P. et al.,
Circulation 75:156-162, 1987; Merlini, P. A. et al., Circulation
90:61-68, 1994; Manten, A. et al., Cardiovasc. Res. 40:389-395,
1998). Furthermore, plasma FPA may indicate the severity of angina
(Gensini, G. F. et al., Thromb. Res. 50:517-525, 1988). Elevations
in the plasma concentration of FPA are associated with any
condition that involves activation of the coagulation pathway,
including stroke, surgery, cancer, disseminated intravascular
coagulation, nephrosis, and thrombotic thrombocytopenic purpura.
FPA is released into the circulation following thrombin activation
and cleavage of fibrinogen. Because FPA is a small polypeptide, it
is likely cleared from the bloodstream rapidly. FPA has been
demonstrated to be elevated for more than one month following clot
formation, and maximum plasma FPA concentrations can exceed 40
ng/ml in active angina (Gensini, G. F. et al., Thromb. Res.
50:517-525, 1988; Tohgi, H. et al., Stroke 21:1663-1667, 1990).
[0147] Platelet-derived growth factor (PDGF) is a 28 kDa secreted
homo- or heterodimeric protein composed of the homologous subunits
A and/or B (Mahadevan, D. et al., J. Biol. Chem. 270:27595-27600,
1995). PDGF is a potent mitogen for mesenchymal cells, and has been
implicated in the pathogenesis of atherosclerosis. PDGF is released
by aggregating platelets and monocytes near sites of vascular
injury. The normal plasma concentration of PDGF is <0.4 ng/ml
(15 pM). Plasma PDGF concentrations are higher in individuals with
acute myocardial infarction and unstable angina than in healthy
controls or individuals with stable angina (Ogawa, H. et al., Am.
J. Cardiol. 69:453-456, 1992; Wallace, J. M. et al., Ann. Clin.
Biochem. 35:236-241, 1998; Ogawa, H. et al., Coron. Artery Dis.
4:437-442, 1993). Changes in the plasma PDGF concentration in these
individuals is most likely due to increased platelet and monocyte
activation. Plasma PDGF is elevated in individuals with brain
tumors, breast cancer, and hypertension (Kurimoto, M. et al., Acta
Neurochir. (Wien) 137:182-187, 1995; Seymour, L. et al., Breast
Cancer Res. Treat. 26:247-252, 1993; Rossi, E. et al., Am. J.
Hypertens. 11:1239-1243, 1998). Plasma PDGF may also be elevated in
any pro-inflammatory condition or any condition that causes
platelet activation including surgery, trauma, disseminated
intravascular coagulation, and thrombotic thrombocytopenic purpura.
PDGF is released from the secretory granules of platelets and
monocytes upon activation. PDGF has a biphasic half-life of
approximately 5 minutes and 1 hour in animals (Cohen, A. M. et al.,
J. Surg. Res. 49:447-452, 1990; Bowen-Pope, D. F. et al., Blood
64:458-469, 1984). The plasma PDGF concentration in ACS can exceed
0.6 ng/ml (22 pM) (Ogawa, H. et al., Am. J. Cardiol. 69:453-456,
1992). PDGF may be a sensitive and specific marker of platelet
activation. In addition, it may be a sensitive marker of vascular
injury, and the accompanying monocyte and platelet activation.
[0148] Prothrombin fragment 1+2 is a 32 kDa polypeptide that is
liberated from the amino terminus of thrombin during thrombin
activation. The normal plasma concentration of F1+2 is <32 ng/ml
(1 nM). The plasma concentration of F1+2 is reportedly elevated in
patients with acute myocardial infarction and unstable angina, but
not stable angina, but the changes were not robust (Merlini, P. A.
et al., Circulation 90:61-68, 1994). Other reports have indicated
that there is no significant change in the plasma F1+2
concentration in cardiovascular disease (Biasucci, L. M. et al.,
Circulation 93:2121-2127, 1996; Manten, A. et al., Cardiovasc. Res.
40:389-395, 1998). The concentration of F1+2 in plasma can be
elevated during any condition associated with coagulation
activation, including stroke, surgery, trauma, thrombotic
thrombocytopenic purpura, and disseminated intravascular
coagulation. F1+2 is released into the bloodstream immediately upon
thrombin activation. F1+2 has a half-life of approximately 90
minutes in plasma, and it has been suggested that this long
half-life may mask bursts of thrombin formation (Biasucci, L. M. et
al., Circulation 93:2121-2127, 1996).
[0149] P-selectin, also called granule membrane protein-140,
GMP-140, PADGEM, and CD-62P, is a .about.140 kDa adhesion molecule
expressed in platelets and endothelial cells. P-selectin is stored
in the alpha granules of platelets and in the Weibel-Palade bodies
of endothelial cells. Upon activation, P-selectin is rapidly
translocated to the surface of endothelial cells and platelets to
facilitate the "rolling" cell surface interaction with neutrophils
and monocytes. Membrane-bound and soluble forms of P-selectin have
been identified. Soluble P-selectin may be produced by shedding of
membrane-bound P-selectin, either by proteolysis of the
extracellular P-selectin molecule, or by proteolysis of components
of the intracellular cytoskeleton in close proximity to the
surface-bound P-selectin molecule (Fox, J. E., Blood Coagul.
Fibrinolysis 5:291-304, 1994). Additionally, soluble P-selectin may
be translated from mRNA that does not encode the N-terminal
transmembrane domain (Dunlop, L. C. et al., J. Exp. Med.
175:1147-1150, 1992; Johnston, G. I. et al., J. Biol. Chem.
265:21381-21385, 1990). Activated platelets can shed membrane-bound
P-selectin and remain in the circulation, and the shedding of
P-selectin can elevate the plasma P-selectin concentration by
approximately 70 ng/ml (Michelson, A. D. et al., Proc. Natl. Acad.
Sci. U.S.A. 93:11877-11882, 1996). Soluble P-selectin may also
adopt a different conformation than membrane-bound P-selectin.
Soluble P-selectin has a monomeric rod-like structure with a
globular domain at one end, and the membrane-bound molecule forms
rosette structures with the globular domain facing outward
(Ushiyama, S. et al., J. Biol. Chem. 268:15229-15237, 1993).
Soluble P-selectin may play an important role in regulating
inflammation and thrombosis by blocking interactions between
leukocytes and activated platelets and endothelial cells (Gamble,
J. R. et al., Science 249:414-417, 1990). The normal plasma
concentration of soluble P-selectin is <200 ng/ml. Blood is
normally collected using citrate as an anticoagulant, but some
studies have used EDTA plasma with additives such as prostaglandin
E to prevent platelet activation. EDTA may be a suitable
anticoagulant that will yield results comparable to those obtained
using citrate. Furthermore, the plasma concentration of soluble
P-selectin may not be affected by potential platelet activation
during the sampling procedure. The plasma soluble P-selectin
concentration was significantly elevated in patients with acute
myocardial infarction and unstable angina, but not stable angina,
even following an exercise stress test (Ikeda, H. et al.,
Circulation 92:1693-1696, 1995; Tomoda, H. and Aoki, N., Angiology
49:807-813, 1998; Hollander, J. E. et al., J. Am. Coll. Cardiol.
34:95-105, 1999; Kaikita, K. et al., Circulation 92:1726-1730,
1995; Ikeda, H. et al., Coron. Artery. Dis. 5:515-518, 1994). The
sensitivity and specificity of membrane-bound P-selectin versus
soluble P-selectin for acute myocardial infarction is 71% versus
76% and 32% versus 45% (Hollander, J. E. et al., J. Am. Coll.
Cardiol. 34:95-105, 1999). The sensitivity and specificity of
membrane-bound P-selectin versus soluble P-selectin for unstable
angina+acute myocardial infarction is 71% versus 79% and 30% versus
35% (Hollander, J. E. et al., J. Am. Coll. Cardiol. 34:95-105,
1999). P-selectin expression is greater in coronary atherectomy
specimens from individuals with unstable angina than stable angina
(Tenaglia, A. N. et al., Am. J. Cardiol. 79:742-747, 1997).
Furthermore, plasma soluble P-selectin may be elevated to a greater
degree in patients with acute myocardial infarction than in
patients with unstable angina. Plasma soluble and membrane-bound
P-selectin also is elevated in individuals with non-insulin
dependent diabetes mellitus and congestive heart failure (Nomura,
S. et al., Thromb. Haemost. 80:388-392, 1998; O'Connor, C. M. et
al., Am. J. Cardiol. 83:1345-1349, 1999). Soluble P-selectin
concentration is elevated in the plasma of individuals with
idiopathic thrombocytopenic purpura, rheumatoid arthritis,
hypercholesterolemia, acute stroke, atherosclerosis, hypertension,
acute lung injury, connective tissue disease, thrombotic
thrombocytopenic purpura, hemolytic uremic syndrome, disseminated
intravascular coagulation, and chronic renal failure (Katayama, M.
et al., Br. J. Haematol. 84:702-710, 1993; Haznedaroglu, I. C. et
al., Acta Haematol. 101:16-20, 1999; Ertenli, I. et al., J.
Rheumatol. 25:1054-1058, 1998; Davi, G. et al., Circulation
97:953-957, 1998; Frijns, C. J. et al., Stroke 28:2214-2218, 1997;
Blann, A. D. et al., Thromb. Haemost. 77:1077-1080, 1997; Blann, A.
D. et al., J. Hum. Hypertens. 11:607-609, 1997; Sakamaki, F. et
al., A. J. Respir. Crit. Care Med. 151:1821-1826, 1995; Takeda, I.
et al., Int. Arch. Allergy Immunol. 105:128-134, 1994; Chong, B. H.
et al., Blood 83:1535-1541, 1994; Bonomini, M. et al., Nephron
79:399-407, 1998). Additionally, any condition that involves
platelet activation can potentially be a source of plasma
elevations in P-selectin. P-selectin is rapidly presented on the
cell surface following platelet of endothelial cell activation.
Soluble P-selectin that has been translated from an alternative
mRNA lacking a transmembrane domain is also released into the
extracellular space following this activation. Soluble P-selectin
can also be formed by proteolysis involving membrane-bound
P-selectin, either directly or indirectly. Plasma soluble
P-selectin is elevated on admission in patients with acute
myocardial infarction treated with tPA or coronary angioplasty,
with a peak elevation occurring 4 hours after onset (Shimomura, H.
et al., Am. J. Cardiol. 81:397-400, 1998). Plasma soluble
P-selectin was elevated less than one hour following an anginal
attack in patients with unstable angina, and the concentration
decreased with time, approaching baseline more than 5 hours after
attack onset (Ikeda, H. et al., Circulation 92:1693-1696, 1995).
The plasma concentration of soluble P-selectin can approach 1
.mu.g/ml in ACS (Ikeda, H. et al., Coron. Artery Dis. 5:515-518,
1994). Further investigation into the release of soluble P-selectin
into and its removal from the bloodstream need to be conducted.
P-selectin may be a sensitive and specific marker of platelet and
endothelial cell activation, conditions that support thrombus
formation and inflammation. It is not, however, a specific marker
of ACS. When used with another marker that is specific for cardiac
tissue injury, P-selectin may be useful in the discrimination of
unstable angina and acute myocardial infarction from stable angina.
Furthermore, soluble P-selectin may be elevated to a greater degree
in acute myocardial infarction than in unstable angina. P-selectin
normally exists in two forms, membrane-bound and soluble. Published
investigations note that a soluble form of P-selectin is produced
by platelets and endothelial cells, and by shedding of
membrane-bound P-selectin, potentially through a proteolytic
mechanism. Soluble P-selectin may prove to be the most useful
currently identified marker of platelet activation, since its
plasma concentration may not be as influenced by the blood sampling
procedure as other markers of platelet activation, such as PF4 and
.beta.-TG.
[0150] Thrombin is a 37 kDa serine proteinase that proteolytically
cleaves fibrinogen to form fibrin, which is ultimately integrated
into a crosslinked network during clot formation. Antithrombin III
(ATIII) is a 65 kDa serine proteinase inhibitor that is a
physiological regulator of thrombin, factor XIa, factor XIIa, and
factor IXa proteolytic activity. The inhibitory activity of ATIII
is dependent upon the binding of heparin. Heparin enhances the
inhibitory activity of ATIII by 2-3 orders of magnitude, resulting
in almost instantaneous inactivation of proteinases inhibited by
ATIII. ATIII inhibits its target proteinases through the formation
of a covalent 1:1 stoichiometric complex. The normal plasma
concentration of the approximately 100 kDa thrombin-ATIII complex
(TAT) is <5 ng/ml (50 pM). TAT concentration is elevated in
patients with acute myocardial infarction and unstable angina,
especially during spontaneous ischemic episodes (Biasucci, L. M. et
al., Am. J. Cardiol. 77:85-87, 1996; Kienast, J. et al., Thromb.
Haemost. 70:550-553, 1993). Furthermore, TAT may be elevated in the
plasma of individuals with stable angina (Manten, A. et al.,
Cardiovasc. Res. 40:389-395, 1998). Other published reports have
found no significant differences in the concentration of TAT in the
plasma of patients with ACS (Manten, A. et al., Cardiovasc. Res.
40:389-395, 1998; Hoffmeister, H. M. et al., Atherosclerosis
144:151-157, 1999). Further investigation is needed to determine
plasma TAT concentration changes associated with ACS. Elevation of
the plasma TAT concentration is associated with any condition
associated with coagulation activation, including stroke, surgery,
trauma, disseminated intravascular coagulation, and thrombotic
thrombocytopenic purpura. TAT is formed immediately following
thrombin activation in the presence of heparin, which is the
limiting factor in this interaction. TAT has a half-life of
approximately 5 minutes in the bloodstream (Biasucci, L. M. et al.,
Am. J. Cardiol. 77:85-87, 1996). TAT concentration is elevated in,
exhibits a sharp drop after 15 minutes, and returns to baseline
less than 1 hour following coagulation activation. The plasma
concentration of TAT can approach 50 ng/ml in ACS (Biasucci, L. M.
et al., Circulation 93:2121-2127, 1996). TAT is a specific marker
of coagulation activation, specifically, thrombin activation.
[0151] von Willebrand factor (vWF) is a plasma protein produced by
platelets, megakaryocytes, and endothelial cells composed of 220
kDa monomers that associate to form a series of high molecular
weight multimers. These multimers normally range in molecular
weight from 600-20,000 kDa. vWF participates in the coagulation
process by stabilizing circulating coagulation factor VIII and by
mediating platelet adhesion to exposed subendothelium, as well as
to other platelets. The A1 domain of vWF binds to the platelet
glycoprotein Ib-IX-V complex and non-fibrillar collagen type VI,
and the A3 domain binds fibrillar collagen types I and III (Emsley,
J. et al., J. Biol. Chem. 273:10396-10401, 1998). Other domains
present in the vWF molecule include the integrin binding domain,
which mediates platelet-platelet interactions, the the protease
cleavage domain, which appears to be relevant to the pathogenesis
of type 11A von Willebrand disease. The interaction of vWF with
platelets is tightly regulated to avoid interactions between vWF
and platelets in normal physiologic conditions. vWF normally exists
in a globular state, and it undergoes a conformation transition to
an extended chain structure under conditions of high sheer stress,
commonly found at sites of vascular injury. This conformational
change exposes intramolecular domains of the molecule and allows
vWF to interact with platelets. Furthermore, shear stress may cause
vWF release from endothelial cells, making a larger number of vWF
molecules available for interactions with platelets. The
conformational change in vWF can be induced in vitro by the
addition of non-physiological modulators like ristocetin and
botrocetin (Miyata, S. et al., J. Biol. Chem. 271:9046-9053, 1996).
At sites of vascular injury, vWF rapidly associates with collagen
in the subendothelial matrix, and virtually irreversibly binds
platelets, effectively forming a bridge between platelets and the
vascular subendothelium at the site of injury. Evidence also
suggests that a conformational change in vWF may not be required
for its interaction with the subendothelial matrix (Sixma, J. J.
and de Groot, P. G., Mayo Clin. Proc. 66:628-633, 1991). This
suggests that vWF may bind to the exposed subendothelial matrix at
sites of vascular injury, undergo a conformational change because
of the high localized shear stress, and rapidly bind circulating
platelets, which will be integrated into the newly formed thrombus.
Measurement of the total amount of vWF would allow one who is
skilled in the art to identify changes in total vWF concentration
associated with stroke or cardiovascular disease. This measurement
could be performed through the measurement of various forms of the
vWF molecule. Measurement of the A1 domain would allow the
measurement of active vWF in the circulation, indicating that a
pro-coagulant state exists because the A1 domain is accessible for
platelet binding. In this regard, an assay that specifically
measures vWF molecules with both the exposed A1 domain and either
the integrin binding domain or the A3 domain would also allow for
the identification of active vWF that would be available for
mediating platelet-platelet interactions or mediate crosslinking of
platelets to vascular subendothelium, respectively. Measurement of
any of these vWF forms, when used in an assay that employs
antibodies specific for the protease cleavage domain may allow
assays to be used to determine the circulating concentration of
various vWF forms in any individual, regardless of the presence of
von Willebrand disease. The normal plasma concentration of vWF is
5-10 .mu.g/ml, or 60-110% activity, as measured by platelet
aggregation. The measurement of specific forms of vWF may be of
importance in any type of vascular disease, including stroke and
cardiovascular disease. The plasma vWF concentration is reportedly
elevated in individuals with acute myocardial infarction and
unstable angina, but not stable angina (Goto, S. et al.,
Circulation 99:608-613, 1999; Tousoulis, D. et al., Int. J.
Cardiol. 56:259-262, 1996; Yazdani, S. et al., J Am Coll Cardiol
30:1284-1287, 1997; Montalescot, G. et al., Circulation
98:294-299). Furthermore, elevations of the plasma vWF
concentration may be a predictor of adverse clinical outcome in
patients with unstable angina (Montalescot, G. et al., Circulation
98:294-299). vWF concentrations also have been demonstrated to be
elevated in patients with stroke and subarachnoid hemorrhage, and
also appear to be useful in assessing risk of mortality following
stroke (Blann, A. et al., Blood Coagul. Fibrinolysis 10:277-284,
1999; Hirashima, Y. et al. Neurochem Res. 22:1249-1255, 1997;
Catto, A. J. et al., Thromb. Hemost. 77:1104-1108, 1997). The
plasma concentration of vWF may be elevated in conjunction with any
event that is associated with endothelial cell damage or platelet
activation. vWF is present at high concentration in the
bloodstream, and it is released from platelets and endothelial
cells upon activation. vWF would likely have the greatest utility
as a marker of platelet activation or, specifically, conditions
that favor platelet activation and adhesion to sites of vascular
injury. The conformation of vWF is also known to be altered by high
shear stress, as would be associated with a partially stenosed
blood vessel. As the blood flows past a stenosed vessel, it is
subjected to shear stress considerably higher than is encountered
in the circulation of an undiseased individual.
[0152] Tissue factor (TF) is a 45 kDa cell surface protein
expressed in brain, kidney, and heart, and in a transcriptionally
regulated manner on perivascular cells and monocytes. TF forms a
complex with factor VIIa in the presence of Ca.sup.2+ ions, and it
is physiologically active when it is membrane bound. This complex
proteolytically cleaves factor X to form factor Xa. It is normally
sequestered from the bloodstream. Tissue factor can be detected in
the bloodstream in a soluble form, bound to factor VIIa, or in a
complex with factor VIIa, and tissue factor pathway inhibitor that
can also include factor Xa. TF also is expressed on the surface of
macrophages, which are commonly found in atherosclerotic plaques.
The normal serum concentration of TF is <0.2 ng/ml (4.5 pM). The
plasma TF concentration is elevated in patients with ischemic heart
disease (Falciani, M. et al., Thromb. Haemost. 79:495-499, 1998).
TF is elevated in patients with unstable angina and acute
myocardial infarction, but not in patients with stable angina
(Falciani, M. et al., Thromb. Haemost. 79:495-499, 1998; Suefuji,
H. et al., Am. Heart J. 134:253-259, 1997; Misumi, K. et al., Am.
J. Cardiol. 81:22-26, 1998). Furthermore, TF expression on
macrophages and TF activity in atherosclerotic plaques is more
common in unstable angina than stable angina (Soejima, H. et al.,
Circulation 99:2908-2913, 1999; Kaikita, K. et al., Arterioscler.
Thromb. Vasc. Biol. 17:2232-2237, 1997; Ardissino, D. et al.,
Lancet 349:769-771, 1997). The differences in plasma TF
concentration in stable versus unstable angina may not be of
statistical significance. Elevations in the serum concentration of
TF are associated with any condition that causes or is a result of
coagulation activation through the extrinsic pathway. These
conditions can include subarachnoid hemorrhage, disseminated
intravascular coagulation, renal failure, vasculitis, and sickle
cell disease. (Hirashima, Y. et al., Stroke 28:1666-1670, 1997;
Takahashi, H. et al., Am. J. Hematol. 46:333-337, 1994; Koyama, T.
et al., Br. J. Haematol. 87:343-347, 1994). TF is released
immediately when vascular injury is coupled with extravascular cell
injury. TF levels in ischemic heart disease patients can exceed 800
pg/ml within 2 days of onset (Falciani, M. et al., Thromb. Haemost.
79:495-499, 1998. TF levels were decreased in the chronic phase of
acute myocardial infarction, as compared with the chronic phase
(Suefuji, H. et al., Am. Heart J 134:253-259, 1997). TF is a
specific marker for activation of the extrinsic coagulation pathway
and the presence of a general hypercoagulable state. It may be a
sensitive marker of vascular injury resulting from plaque
rupture
[0153] The coagulation cascade can be activated through either the
extrinsic or intrinsic pathways. These enzymatic pathways share one
final common pathway. The first step of the common pathway involves
the proteolytic cleavage of prothrombin by the factor Xa/factor Va
prothrombinase complex to yield active thrombin. Thrombin is a
serine proteinase that proteolytically cleaves fibrinogen. Thrombin
first removes fibrinopeptide A from fibrinogen, yielding desAA
fibrin monomer, which can form complexes with all other
fibrinogen-derived proteins, including fibrin degradation products,
fibrinogen degradation products, desAA fibrin, and fibrinogen. The
desAA fibrin monomer is generically referred to as soluble fibrin,
as it is the first product of fibrinogen cleavage, but it is not
yet crosslinked via factor XIIIa into an insoluble fibrin clot.
DesAA fibrin monomer also can undergo further proteolytic cleavage
by thrombin to remove fibrinopeptide B, yielding desAABB fibrin
monomer. This monomer can polymerize with other desAABB fibrin
monomers to form soluble desAABB fibrin polymer, also referred to
as soluble fibrin or thrombus precursor protein (TpP.TM.). TpP.TM.
is the immediate precursor to insoluble fibrin, which forms a
"mesh-like" structure to provide structural rigidity to the newly
formed thrombus. In this regard, measurement of TpP.TM. in plasma
is a direct measurement of active clot formation. The normal plasma
concentration of TpP.TM. is <6 ng/ml (Laurino, J. P. et al.,
Ann. Clin. Lab. Sci. 27:338-345, 1997). American Biogenetic
Sciences has developed an assay for TpP.TM. (U.S. Pat. Nos.
5,453,359 and 5,843,690) and states that its TpP.TM. assay can
assist in the early diagnosis of acute myocardial infarction, the
ruling out of acute myocardial infarction in chest pain patients,
and the identification of patients with unstable angina that will
progress to acute myocardial infarction. Other studies have
confirmed that TpP.TM. is elevated in patients with acute
myocardial infarction, most often within 6 hours of onset (Laurino,
J. P. et al., Ann. Clin. Lab. Sci. 27:338-345, 1997; Carville, D.
G. et al., Clin. Chem. 42:1537-1541, 1996). The plasma
concentration of TpP.TM. is also elevated in patients with unstable
angina, but these elevations may be indicative of the severity of
angina and the eventual progression to acute myocardial infarction
(Laurino, J. P. et al., Ann. Clin. Lab. Sci. 27:338-345, 1997). The
concentration of TpP.TM. in plasma will theoretically be elevated
during any condition that causes or is a result of coagulation
activation, including disseminated intravascular coagulation, deep
venous thrombosis, congestive heart failure, surgery, cancer,
gastroenteritis, and cocaine overdose (Laurino, J. P. et al., Ann.
Clin. Lab. Sci. 27:338-345, 1997). TpP.TM. is released into the
bloodstream immediately following thrombin activation. TpP.TM.
likely has a short half-life in the bloodstream because it will be
rapidly converted to insoluble fibrin at the site of clot
formation. Plasma TpP.TM. concentrations peak within 3 hours of
acute myocardial infarction onset, returning to normal after 12
hours from onset. The plasma concentration of TpP.TM. can exceed 30
ng/ml in CVD (Laurino, J. P. et al., Ann. Clin. Lab. Sci.
27:338-345, 1997). TpP.TM. is a sensitive and specific marker of
coagulation activation. It has been demonstrated that TpP.TM. is
useful in the diagnosis of acute myocardial infarction, but only
when it is used in conjunction with a specific marker of cardiac
tissue injury.
[0154] (iii) Exemplar Markers Related to Atherosclerotic Plague
Rupture
[0155] The appearance of markers related to atherosclerotic plaque
rupture may preceed specific markers of myocardial injury.
Potential markers of atherosclerotic plaque rupture include human
neutrophil elastase, inducible nitric oxide synthase,
lysophosphatidic acid, malondialdehyde-modified low density
lipoprotein, and various members of the matrix metalloproteinase
(MMP) family, including MMP-1, -2, -3, and -9.
[0156] Human neutrophil elastase (HNE) is a 30 kDa serine
proteinase that is normally contained within the azurophilic
granules of neutrophils. HNE is released upon neutrophil
activation, and its activity is regulated by circulating
.alpha..sub.1-proteinase inhibitor. Activated neutrophils are
commonly found in atherosclerotic plaques, and rupture of these
plaques may result in the release of HNE. The plasma HNE
concentration is usually measured by detecting HNE-.alpha..sub.1-PI
complexes. The normal concentration of these complexes is 50 ng/ml,
which indicates a normal concentration of approximately 25 ng/ml
(0.8 nM) for HNE. HNE release also can be measured through the
specific detection of fibrinopeptide B.beta..sub.30-43, a specific
HNE-derived fibrinopeptide, in plasma. Plasma HNE is elevated in
patients with coronary stenosis, and its elevation is greater in
patients with complex plaques than those with simple plaques
(Kosar, F. et al., Angiology 49:193-201, 1998; Amaro, A. et al.,
Eur. Heart J 16:615-622, 1995). Plasma HNE is not significantly
elevated in patients with stable angina, but is elevated inpatients
with unstable angina and acute myocardial infarction, as determined
by measuring fibrinopeptide B.beta..sub.30-43, with concentrations
in unstable angina being 2.5-fold higher than those associated with
acute myocardial infarction (Dinerman, J. L. et al., J. Am. Coll.
Cardiol. 15:1559-1563, 1990; Mehta, J. et al., Circulation
79:549-556, 1989). Serum HNE is elevated in cardiac surgery,
exercise-induced muscle damage, giant cell arteritis, acute
respiratory distress syndrome, appendicitis, pancreatitis, sepsis,
smoking-associated emphysema, and cystic fibrosis (Genereau, T. et
al., J. Rheumatol. 25:710-713, 1998; Mooser, V. et al.,
Arterioscler. Thromb. Vasc. Biol. 19:1060-1065, 1999; Gleeson, M.
et al. Eur. J. Appl. Physiol. 77:543-546, 1998; Gando, S. et al., J
Trauma 42:1068-1072, 1997; Eriksson, S. et al., Eur. J. Surg.
161:901-905, 1995; Liras, G. et al., Rev. Esp. Enferm. Dig.
87:641-652, 1995; Endo, S. et al., J Inflamm. 45:136-142, 1995;
Janoff, A., Annu Rev Med 36:207-216, 1985). HNE may also be
released during blood coagulation (Plow, E. F. and Plescia, J.,
Thromb. Haemost. 59:360-363, 1988; Plow, E. F., J. Clin. Invest.
69:564-572, 1982). Serum elevations of HNE could also be associated
with any non-specific infection or inflammatory state that involves
neutrophil recruitment and activation. It is most likely released
upon plaque rupture, since activated neutrophils are present in
atherosclerotic plaques. HNE is presumably cleared by the liver
after it has formed a complex with .alpha..sub.1-PI.
[0157] Inducible nitric oxide synthase (iNOS) is a 130 kDa
cytosolic protein in epithelial cells macrophages whose expression
is regulated by cytokines, including interferon-.gamma.,
interleukin-1.beta., interleukin-6, and tumor necrosis factor a,
and lipopolysaccharide. iNOS catalyzes the synthesis of nitric
oxide (NO) from L-arginine, and its induction results in a
sustained high-output production of NO, which has antimicrobial
activity and is a mediator of a variety of physiological and
inflammatory events. NO production by iNOS is approximately 100
fold more than the amount produced by constitutively-expressed NOS
(Depre, C. et al., Cardiovasc. Res. 41:465-472, 1999). There are no
published investigations of plasma iNOS concentration changes
associated with ACS. iNOS is expressed in coronary atherosclerotic
plaque, and it may interfere with plaque stability through the
production of peroxynitrate, which is a product of NO and
superoxide and enhances platelet adhesion and aggregation (Depre,
C. et al., Cardiovasc. Res. 41:465-472, 1999). iNOS expression
during myocardial ischemia may not be elevated, suggesting that
iNOS may be useful in the differentiation of angina from acute
myocardial infarction (Hammerman, S. I. et al., Am. J. Physiol.
277:H1579-H1592, 1999; Kaye, D. M. et al., Life Sci 62:883-887,
1998). Elevations in the plasma iNOS concentration may be
associated with cirrhosis, iron-deficiency anemia, or any other
condition that results in macrophage activation, including
bacterial infection (Jimenez, W. et al., Hepatology 30:670-676,
1999; Ni, Z. et al., Kidney Int. 52:195-201, 1997). iNOS may be
released into the bloodstream as a result of atherosclerotic plaque
rupture, and the presence of increased amounts of iNOS in the
bloodstream may not only indicate that plaque rupture has occurred,
but also that an ideal environment has been created to promote
platelet adhesion. However, iNOS is not specific for
atherosclerotic plaque rupture, and its expression can be induced
during non-specific inflammatory conditions.
[0158] Lysophosphatidic acid (LPA) is a lysophospholipid
intermediate formed in the synthesis of phosphoglycerides and
triacylglycerols. It is formed by the acylation of glycerol-3
phosphate by acyl-coenzyme A and during mild oxidation of
low-density lipoprotein (LDL). LPA is a lipid second messanger with
vasoactive properties, and it can function as a platelet activator.
LPA is a component of atherosclerotic lesions, particularly in the
core, which is most prone to rupture (Siess, W., Proc. Natl. Acad.
Sci. U.S.A. 96, 6931-6936, 1999). The normal plasma LPA
concentration is 540 nM. Serum LPA is elevated in renal failure and
in ovarian cancer and other gynecologic cancers (Sasagawa, T. et
al., J. Nutr. Sci. Vitaminol. (Tokyo) 44:809-818, 1998; Xu, Y. et
al., JAMA 280:719-723, 1998). In the context of unstable angina,
LPA is most likely released as a direct result of plaque rupture.
The plasma LPA concentration can exceed 60 .mu.M in patients with
gynecologic cancers (Xu, Y. et al., JAMA 280:719-723, 1998). Serum
LPA may be a useful marker of atherosclerotic plaque rupture.
[0159] Malondialdehyde-modified low-density lipoprotein
(MDA-modified LDL) is formed during the oxidation of the apoB-100
moiety of LDL as a result of phospholipase activity, prostaglandin
synthesis, or platelet activation. MDA-modified LDL can be
distinguished from oxidized LDL because MDA modifications of LDL
occur in the absence of lipid peroxidation (Holvoet, P., Acta
Cardiol. 53:253-260, 1998). The normal plasma concentration of
MDA-modified LDL is less than 4 .mu.g/ml (.about.10 .mu.M). Plasma
concentrations of oxidized LDL are elevated in stable angina,
unstable angina, and acute myocardial infarction, indicating that
it may be a marker of atherosclerosis (Holvoet, P., Acta Cardiol.
53:253-260, 1998; Holvoet, P. et al., Circulation 98:1487-1494,
1998). Plasma MDA-modified LDL is not elevated in stable angina,
but is significantly elevated in unstable angina and acute
myocardial infarction (Holvoet, P., Acta Cardiol. 53:253-260, 1998;
Holvoet, P. et al., Circulation 98:1487-1494, 1998; Holvoet, P. et
al., JAMA 281:1718-1721, 1999). Plasma MDA-modified LDL is elevated
in individuals with beta-thallasemia and in renal transplant
patients (Livrea, M. A. et al., Blood 92:3936-3942, 1998; Ghanem,
H. et al., Kidney Int. 49:488-493, 1996; van den Dorpel, M. A. et
al., Transpl. Int. 9 Suppl. 1:S54-S57, 1996). Furthermore, serum
MDA-modified LDL may be elevated during hypoxia (Balagopalakrishna,
C. et al., Adv. Exp. Med. Biol. 411:337-345, 1997). The plasma
concentration of MDA-modified LDL is elevated within 6-8 hours from
the onset of chest pain. Plasma concentrations of MDA-modified LDL
can approach 20 .mu.g/ml (.about.50 .mu.M) in patients with acute
myocardial infarction, and 15 .mu.g/ml (.about.40 .mu.M) in
patients with unstable angina (Holvoet, P. et al., Circulation
98:1487-1494, 1998). Plasma MDA-modified LDL has a half-life of
less than 5 minutes in mice (Ling, W. et al., J. Clin. Invest.
100:244-252, 1997). MDA-modified LDL appears to be a specific
marker of atherosclerotic plaque rupture in acute coronary
symptoms. It is unclear, however, if elevations in the plasma
concentration of MDA-modified LDL are a result of plaque rupture or
platelet activation. The most reasonable explanation is that the
presence of increased amounts of MDA-modified LDL is an indication
of both events. MDA-modified LDL may be useful in discriminating
unstable angina and acute myocardial infarction from stable
angina.
[0160] Matrix metalloproteinase-1 (MMP-1), also called
collagenase-1, is a 41/44 kDa zinc- and calcium-binding proteinase
that cleaves primarily type I collagen, but can also cleave
collagen types II, III, VII and X. The active 41/44 kDa enzyme can
undergo autolysis to the still active 22/27 kDa form. MMP-1 is
synthesized by a variety of cells, including smooth muscle cells,
mast cells, macrophage-derived foam cells, T lymphocytes, and
endothelial cells (Johnson, J. L. et al., Arterioscler. Thromb.
Vasc. Biol. 18:1707-1715, 1998). MMP-1, like other MMPs, is
involved in extracellular matrix remodeling, which can occur
following injury or during intervascular cell migration. MMP-1 can
be found in the bloodstream either in a free form or in complex
with TIMP-1, its natural inhibitor. MMP-1 is normally found at a
concentration of <25 ng/ml in plasma. MMP-1 is found in the
shoulder region of atherosclerotic plaques, which is the region
most prone to rupture, and may be involved in atherosclerotic
plaque destabilization (Johnson, J. L. et al., Arterioscler.
Thromb. Vasc. Biol. 18:1707-1715, 1998). Furthermore, MMP-1 has
been implicated in the pathogenesis of myocardial reperfusion
injury (Shibata, M. et al., Angiology 50:573-582, 1999). Serum
MMP-1 may be elevated inflammatory conditions that induce mast cell
degranulation. Serum MMP-1 concentrations are elevated in patients
with arthritis and systemic lupus erythematosus (Keyszer, G. et
al., Z Rheumatol 57:392-398, 1998; Keyszer, G. J. Rheumatol.
26:251-258, 1999). Serum MMP-1 also is elevated in patients with
prostate cancer, and the degree of elevation corresponds to the
metastatic potential of the tumor (Baker, T. et al., Br. J. Cancer
70:506-512, 1994). The serum concentration of MMP-1 may also be
elevated in patients with other types of cancer. Serum MMP-1 is
decreased in patients with hemochromatosis and also in patients
with chronic viral hepatitis, where the concentration is inversely
related to the severity (George, D. K. et al., Gut 42:715-720,
1998; Murawaki, Y. et al., J. Gastroenterol. Hepatol. 14:138-145,
1999). Serum MMP-1 was decreased in the first four days following
acute myocardial infarction, and increased thereafter, reaching
peak levels 2 weeks after the onset of acute myocardial infarction
(George, D. K. et al., Gut 42:715-720, 1998).
[0161] Matrix metalloproteinase-2 (MMP-2), also called gelatinase
A, is a 66 kDa zinc- and calcium-binding proteinase that is
synthesized as an inactive 72 kDa precursor. Mature MMP-3 cleaves
type I gelatin and collagen of types IV, V, VII, and X. MMP-2 is
synthesized by a variety of cells, including vascular smooth muscle
cells, mast cells, macrophage-derived foam cells, T lymphocytes,
and endothelial cells (Johnson, J. L. et al., Arterioscler. Thromb.
Vasc. Biol. 18:1707-1715, 1998). MMP-2 is usually found in plasma
in complex with TIMP-2, its physiological regulator (Murawaki, Y.
et al., J. Hepatol. 30:1090-1098, 1999). The normal plasma
concentration of MMP-2 is <.about.550 ng/ml (8 nM). MMP-2
expression is elevated in vascular smooth muscle cells within
atherosclerotic lesions, and it may be released into the
bloodstream in cases of plaque instability (Kai, H. et al., J. Am.
Coll. Cardiol. 32:368-372, 1998). Furthermore, MMP-2 has been
implicated as a contributor to plaque instability and rupture
(Shah, P. K. et al., Circulation 92:1565-1569, 1995). Serum MMP-2
concentrations were elevated in patients with stable angina,
unstable angina, and acute myocardial infarction, with elevations
being significantly greater in unstable angina and acute myocardial
infarction than in stable angina (Kai, H. et al., J. Am. Coll.
Cardiol. 32:368-372, 1998). There was no change in the serum MMP-2
concentration in individuals with stable angina following a
treadmill exercise test (Kai, H. et al., J. Am. Coll. Cardiol.
32:368-372, 1998). Serum and plasma MMP-2 is elevated in patients
with gastric cancer, hepatocellular carcinoma, liver cirrhosis,
urothelial carcinoma, rheumatoid arthritis, and lung cancer
(Murawaki, Y. et al., J. Hepatol. 30:1090-1098, 1999; Endo, K. et
al., Anticancer Res. 17:2253-2258, 1997; Gohji, K. et al., Cancer
78:2379-2387, 1996; Gruber, B. L. et al., Clin. Immunol.
Immunopathol. 78:161-171, 1996; Garbisa, S. et al., Cancer Res.
52:4548-4549, 1992). Furthermore, MMP-2 may also be translocated
from the platelet cytosol to the extracellular space during
platelet aggregation (Sawicki, G. et al., Thromb. Haemost.
80:836-839, 1998). MMP-2 was elevated on admission in the serum of
individuals with unstable angina and acute myocardial infarction,
with maximum levels approaching 1.5 .mu.g/ml (25 nM) (Kai, H. et
al., J. Am. Coll. Cardiol. 32:368-372, 1998). The serum MMP-2
concentration peaked 1-3 days after onset in both unstable angina
and acute myocardial infarction, and started to return to normal
after 1 week (Kai, H. et al., J. Am. Coll. Cardiol. 32:368-372,
1998).
[0162] Matrix metalloproteinase-3 (MMP-3), also called
stromelysin-1, is a 45 kDa zinc- and calcium-binding proteinase
that is synthesized as an inactive 60 kDa precursor. Mature MMP-3
cleaves proteoglycan, fibrinectin, laminin, and type IV collagen,
but not type I collagen. MMP-3 is synthesized by a variety of
cells, including smooth muscle cells, mast cells,
macrophage-derived foam cells, T lymphocytes, and endothelial cells
(Johnson, J. L. et al., Arterioscler. Thromb. Vasc. Biol.
18:1707-1715, 1998). MMP-3, like other MMPs, is involved in
extracellular matrix remodeling, which can occur following injury
or during intervascular cell migration. MMP-3 is normally found at
a concentration of <125 ng/ml in plasma. The serum MMP-3
concentration also has been shown to increase with age, and the
concentration in males is approximately 2 times higher in males
than in females (Manicourt, D. H. et al., Arthritis Rheum.
37:1774-1783, 1994). MMP-3 is found in the shoulder region of
atherosclerotic plaques, which is the region most prone to rupture,
and may be involved in atherosclerotic plaque destabilization
(Johnson, J. L. et al., Arterioscler. Thromb. Vasc. Biol.
18:1707-1715, 1998). Therefore, MMP-3 concentration may be elevated
as a result of atherosclerotic plaque rupture in unstable angina.
Serum MMP-3 may be elevated inflammatory conditions that induce
mast cell degranulation. Serum MMP-3 concentrations are elevated in
patients with arthritis and systemic lupus erythematosus (Zucker,
S. et al. J. Rheumatol. 26:78-80, 1999; Keyszer, G. et al., Z
Rheumatol. 57:392-398, 1998; Keyszer, G. et al. J. Rheumatol.
26:251-258, 1999). Serum MMP-3 also is elevated in patients with
prostate and urothelial cancer, and also glomerulonephritis (Lein,
M. et al., Urologe A 37:377-381, 1998; Gohji, K. et al., Cancer
78:2379-2387, 1996; Akiyama, K. et al., Res. Commun. Mol. Pathol.
Pharmacol. 95:115-128, 1997). The serum concentration of MMP-3 may
also be elevated in patients with other types of cancer. Serum
MMP-3 is decreased in patients with hemochromatosis (George, D. K.
et al., Gut 42:715-720, 1998).
[0163] Matrix metalloproteinase-9 (MMP-9) also called gelatinase B,
is an 84 kDa zinc- and calcium-binding proteinase that is
synthesized as an inactive 92 kDa precursor. Mature MMP-9 cleaves
gelatin types I and V, and collagen types IV and V. MMP-9 exists as
a monomer, a homodimer, and a heterodimer with a 25 kDa
.alpha..sub.2-microglobulin-related protein (Triebel, S. et al.,
FEBS Lett. 314:386-388, 1992). MMP-9 is synthesized by a variety of
cell types, most notably by neutrophils. The normal plasma
concentration of MMP-9 is <35 ng/ml (400 pM). MMP-9 expression
is elevated in vascular smooth muscle cells within atherosclerotic
lesions, and it may be released into the bloodstream in cases of
plaque instability (Kai, H. et al., J. Am. Coll. Cardiol.
32:368-372, 1998). Furthermore, MMP-9 may have a pathogenic role in
the development of ACS (Brown, D. L. et al., Circulation
91:2125-2131, 1995). Plasma MMP-9 concentrations are significantly
elevated in patients with unstable angina and acute myocardial
infarction, but not stable angina (Kai, H. et al., J. Am. Coll.
Cardiol. 32:368-372, 1998). The elevations in patients with acute
myocardial infarction may also indicate that those individuals were
suffering from unstable angina. Elevations in the plasma
concentration of MMP-9 may also be greater in unstable angina than
in acute myocardial infarction. There was no significant change in
plasma MMP-9 levels after a treadmill exercise test in patients
with stable angina (Kai, H. et al., J. Am. Coll. Cardiol.
32:368-372, 1998). Plasma MMP-9 is elevated in individuals with
rheumatoid arthritis, septic shock, giant cell arteritis and
various carcinomas (Gruber, B. L. et al., Clin. Immunol.
Immunopathol. 78:161-171, 1996; Nakamura, T. et al., Am. J. Med.
Sci. 316:355-360, 1998; Blankaert, D. et al., J. Acquir. Immune
Defic. Syndr. Hum. Retrovirol. 18:203-209, 1998; Endo, K. et al.
Anticancer Res. 17:2253-2258, 1997; Hayasaka, A. et al., Hepatology
24:1058-1062, 1996; Moore, D. H. et al., Gynecol. Oncol. 65:78-82,
1997; Sorbi, D. et al., Arthritis Rheum. 39:1747-1753, 1996;
lizasa, T. et al., Clin., Cancer Res. 5:149-153, 1999).
Furthermore, the plasma MMP-9 concentration may be elevated in
stroke and cerebral hemorrhage (Mun-Bryce, S. and Rosenberg, G. A.,
J. Cereb. Blood Flow Metab. 18:1163-1172, 1998; Romanic, A. M. et
al., Stroke 29:1020-1030, 1998; Rosenberg, G. A., J. Neurotrauma
12:833-842, 1995). MMP-9 was elevated on admission in the serum of
individuals with unstable angina and acute myocardial infarction,
with maximum levels approaching 150 ng/ml (1.7 nM) (Kai, H. et al.,
J. Am. Coll. Cardiol. 32:368-372, 1998). The serum MMP-9
concentration was highest on admission in patients unstable angina,
and the concentration decreased gradually after treatment,
approaching baseline more than 1 week after onset (Kai, H. et al.,
J. Am. Coll. Cardiol. 32:368-372, 1998).
[0164] The balance between matrix metalloproteinases and their
inhibitors is a critical factor which affects tumor invasion and
metastasis. The TIMP family represents a class of small (21-28 kDa)
related proteins that inhibit the metalloproteinases. Tissue
inhibitor of metalloproteinase 1 (TIMP 1) is reportedly involved in
the regulation of bone modeling and remodeling in normal developing
human bone, involved in the invasive phenotype of acute myelogenous
leukemia, demonstrating polymorphic X-chromosome inactivation.
TIMP1 is known to act on mmp-1, mmp-2, mmp-3, mmp-7, mmp-8, mmp-9,
mmp-10, mmp-11, mmp-12, mmp-13 and mmp-16. Tissue inhibitor of
metalloproteinase 2 (TIMP2) complexes with metalloproteinases (such
as collagenases) and irreversibly inactivates them. TIMP 2 is known
to act on mmp-1, mmp-2, mmp-3, mmp-7, mmp-8, mmp-9, mmp-10, mmp-13,
mmp-14, mmp-15, mmp-16 and mmp-19. Two alternatively spliced forms
may be associated with SYN4, and involved in the invasive phenotype
of acute myelogenous leukemia. Unlike the inducible expression of
some other TIMP gene family members, the expression of this gene is
largely constitutive. Tissue inhibitor of metalloproteinase 3
(TIMP3) antagonizes matrix metalloproteinase activity and can
suppress tumor growth, angiogenesis, invasion, and metastasis. Loss
of TIMP-3 has been related to the acquisition of tumorigenesis.
[0165] (iv) Exemplary Markers Related to Tissue Injury and
Inflammation
[0166] Pulmonary surfactant protein D (SP-D) is a 43 kDa protein
synthesized and secreted into the airspaces of the lung by the
respiratory epithelium. At the alveolar level, SP-D is
constitutively synthesized and secreted by alveolar type II cells.
SP-D, a collagenous calcium-dependent lectin (or collectin), binds
to surface glycoconjugates expressed by a wide variety of
microorganisms, and to oligosaccharides associated with the surface
of various complex organic antigens. SP-D also specifically
interacts with glycoconjugates and other molecules expressed on the
surface of macrophages, neutrophils, and lymphocytes. In addition,
SP-D binds to specific surfactant-associated lipids and can
influence the organization of lipid mixtures containing
phosphatidylinositol in vitro. Consistent with these diverse in
vitro activities is the observation that SP-D-deficient transgenic
mice show abnormal accumulations of surfactant lipids, and respond
abnormally to challenge with respiratory viruses and bacterial
lipopolysaccharides. The phenotype of macrophages isolated from the
lungs of SP-D-deficient mice is altered, and there is
circumstantial evidence that abnormal oxidant metabolism and/or
increased metalloproteinase expression contributes to the
development of emphysema. The expression of SP-D is increased in
response to many forms of lung injury, and deficient accumulation
of appropriately oligomerized SP-D might contribute to the
pathogenesis of a variety of human lung diseases. See, e.g.,
Crouch, Respir. Res. 1: 93-108 (2000).
[0167] C-reactive protein is a (CRP) is a homopentameric
Ca.sup.2+-binding acute phase protein with 21 kDa subunits that is
involved in host defense. CRP preferentially binds to
phosphorylcholine, a common constituent of microbial membranes.
Phosphorylcholine is also found in mammalian cell membranes, but it
is not present in a form that is reactive with CRP. The interaction
of CRP with phosphorylcholine promotes agglutination and
opsonization of bacteria, as well as activation of the complement
cascade, all of which are involved in bacterial clearance.
Furthermore, CRP can interact with DNA and histones, and it has
been suggested that CRP is a scavenger of nuclear material released
from damaged cells into the circulation (Robey, F. A. et al., J.
Biol. Chem. 259:7311-7316, 1984). CRP synthesis is induced by 11-6,
and indirectly by IL-1, since IL-1 can trigger the synthesis of
IL-6 by Kupffer cells in the hepatic sinusoids. The normal plasma
concentration of CRP is <3 .mu.g/ml (30 nM) in 90% of the
healthy population, and <10 .mu.g/ml (100 nM) in 99% of healthy
individuals. Plasma CRP concentrations can be measured by rate
nephelometry or ELISA. The plasma concentration of CRP is
significantly elevated in patients with acute myocardial infarction
and unstable angina, but not stable angina (Biasucci, L. M. et al.,
Circulation 94:874-877, 1996; Biasucci, L. M. et al., Am. J.
Cardiol. 77:85-87, 1996; Benamer, H. et al., Am. J. Cardiol.
82:845-850, 1998; Caligiuri, G. et al., J. Am. Coll. Cardiol.
32:1295-1304, 1998; Curzen, N. P. et al., Heart 80:23-27, 1998;
Dangas, G. et al., Am. J. Cardiol. 83:583-5, A7, 1999). CRP may
also be elevated in the plasma of individuals with variant or
resolving unstable angina, but mixed results have been reported
(Benamer, H. et al., Am. J. Cardiol. 82:845-850, 1998; Caligiuri,
G. et al., J. Am. Coll. Cardiol. 32:1295-1304, 1998). The
concentration of CRP will be elevated in the plasma from
individuals with any condition that may elicit an acute phase
response, such as infection, surgery, trauma, and stroke. CRP is a
secreted protein that is released into the bloodstream soon after
synthesis. CRP synthesis is upregulated by IL-6, and the plasma CRP
concentration is significantly elevated within 6 hours of
stimulation (Biasucci, L. M. et al., Am. J. Cardiol. 77:85-87,
1996). The plasma CRP concentration peaks approximately 50 hours
after stimulation, and begins to decrease with a half-life of
approximately 19 hours in the bloodstream (Biasucci, L. M. et al.,
Am. J. Cardiol. 77:85-87, 1996). Other investigations have
confirmed that the plasma CRP concentration in individuals with
unstable angina (Biasucci, L. M. et al., Circulation 94:874-877,
1996). The plasma concentration of CRP can approach 100 .mu.g/ml (1
.mu.M) in individuals with ACS (Biasucci, L. M. et al., Circulation
94:874-877, 1996; Liuzzo, G. et al., Circulation 94:2373-2380,
1996). CRP is a specific marker of the acute phase response.
Elevations of CRP have been identified in the plasma of individuals
with acute myocardial infarction and unstable angina, most likely
as a result of activation of the acute phase response associated
with atherosclerotic plaque rupture or cardiac tissue injury.
[0168] Interleukins (ILs) are part of a larger class of
polypeptides known as cytokines. These are messenger molecules that
transmit signals between various cells of the immune system. They
are mostly secreted by macrophages and lymphocytes and their
production is induced in response to injury or infection. Their
actions influence other cells of the immune system as well as other
tissues and organs including the liver and brain. There are 18 ILs
described up till now. IL-2, IL-4, IL-6, IL-8 and IL-10 are the
important interleukins. The following table shows selected
functions of representative interleukins.
3TABLE 1 Selected Functions of Representative Interleukins*
Functions IL-1 IL-2 IL-4 IL-6 IL-8 IL-10 Enhance immune responses +
+ + + - + Suppress immune responses - - - - - + Enhance
inflammation + + + + + - Suppress inflammation - - - - - + Promote
cell growth + + - - - - Chemotactic (chemokines) - - - - + -
Pyrogenic + - - - - -
[0169] Interleukin-1.beta. (IL-1.beta.) is a 17 kDa secreted
proinflammatory cytokine that is involved in the acute phase
response and is a pathogenic mediator of many diseases. IL-1.beta.
is normally produced by macrophages and epithelial cells.
IL-1.beta. is also released from cells undergoing apoptosis. The
normal serum concentration of IL-1.beta. is <30 pg/ml (1.8 pM).
In theory, IL-1.beta. would be elevated earlier than other acute
phase proteins such as CRP in unstable angina and acute myocardial
infarction, since IL-1.beta. is an early participant in the acute
phase response. Furthermore, IL-1.beta. is released from cells
undergoing apoptosis, which may be activated in the early stages of
ischemia. In this regard, elevation of the plasma IL-1.beta.
concentration associated with ACS requires further investigation
using a high-sensitivity assay. Elevations of the plasma IL-1.beta.
concentration are associated with activation of the acute phase
response in proinflammatory conditions such as trauma and
infection. IL-1.beta. has a biphasic physiological half-life of 5
minutes followed by 4 hours (Kudo, S. et al., Cancer Res.
50:5751-5755, 1990). IL-1.beta. is released into the extracellular
milieu upon activation of the inflammatory response or
apoptosis.
[0170] Interleukin-1 receptor antagonist (IL-1ra) is a 17 kDa
member of the IL-1 family predominantly expressed in hepatocytes,
epithelial cells, monocytes, macrophages, and neutrophils. IL-1ra
has both intracellular and extracellular forms produced through
alternative splicing. IL-1ra is thought to participate in the
regulation of physiological IL-1 activity. IL-1ra has no IL-1-like
physiological activity, but is able to bind the IL-1 receptor on
T-cells and fibroblasts with an affinity similar to that of
IL-1.beta., blocking the binding of IL-1.alpha. and IL-1.beta. and
inhibiting their bioactivity (Stockman, B. J. et al., Biochemistry
31:5237-5245, 1992; Eisenberg, S. P. et al., Proc. Natl. Acad. Sci.
U.S.A. 88:5232-5236, 1991; Carter, D. B. et al., Nature
344:633-638, 1990). IL-1ra is normally present in higher
concentrations than IL-1 in plasma, and it has been suggested that
IL-1ra levels are a better correlate of disease severity than IL-1
(Biasucci, L. M. et al., Circulation 99:2079-2084, 1999).
Furthermore, there is evidence that IL-1ra is an acute phase
protein (Gabay, C. et al., J. Clin. Invest. 99:2930-2940, 1997).
The normal plasma concentration of IL-Ira is <200 pg/ml (12 pM).
The plasma concentration of IL-1ra is elevated in patients with
acute myocardial infarction and unstable angina that proceeded to
acute myocardial infarction, death, or refractory angina (Biasucci,
L. M. et al., Circulation 99:2079-2084, 1999; Latini, R. et al., J.
Cardiovasc. Pharmacol. 23:1-6, 1994). Furthermore, IL-1ra was
significantly elevated in severe acute myocardial infarction as
compared to uncomplicated acute myocardial infarction (Latini, R.
et al., J. Cardiovasc. Pharmacol. 23:1-6, 1994). Elevations in the
plasma concentration of IL-1ra are associated with any condition
that involves activation of the inflammatory or acute phase
response, including infection, trauma, and arthritis. IL-1ra is
released into the bloodstream in pro-inflammatory conditions, and
it may also be released as a participant in the acute phase
response. The major sources of clearance of IL-1ra from the
bloodstream appear to be kidney and liver (Kim, D. C. et al., J.
Pharm. Sci. 84:575-580, 1995). IL-1ra concentrations were elevated
in the plasma of individuals with unstable angina within 24 hours
of onset, and these elevations may even be evident within 2 hours
of onset (Biasucci, L. M. et al., Circulation 99:2079-2084, 1999).
In patients with severe progression of unstable angina, the plasma
concentration of IL-1ra was higher 48 hours after onset than levels
at admission, while the concentration decreased in patients with
uneventful progression (Biasucci, L. M. et al., Circulation
99:2079-2084, 1999). In addition, the plasma concentration of
IL-1ra associated with unstable angina can approach 1.4 ng/ml (80
pM). Changes in the plasma concentration of IL-1ra appear to be
related to disease severity. Furthermore, it is likely released in
conjunction with or soon after IL-1 release in pro-inflammatory
conditions, and it is found at higher concentrations than IL-1.
This indicates that IL-1ra may be a useful indirect marker of IL-1
activity, which elicits the production of IL-6.
[0171] Interleukin-6 (IL-6) is a 20 kDa secreted protein that is a
hematopoietin family proinflammatory cytokine. IL-6 is an
acute-phase reactant and stimulates the synthesis of a variety of
proteins, including adhesion molecules. Its major function is to
mediate the acute phase production of hepatic proteins, and its
synthesis is induced by the cytokine IL-1. IL-6 is normally
produced by macrophages and T lymphocytes. The normal serum
concentration of IL-6 is <3 pg/ml (0.15 pM). The plasma
concentration of IL-6 is elevated in patients with acute myocardial
infarction and unstable angina, to a greater degree in acute
myocardial infarction (Biasucci, L. M. et al., Circulation
94:874-877, 1996; Manten, A. et al., Cardiovasc. Res. 40:389-395,
1998; Biasucci, L. M. et al., Circulation 99:2079-2084, 1999). IL-6
is not significantly elevated in the plasma of patients with stable
angina (Biasucci, L. M. et al., Circulation 94:874-877, 1996;
Manten, A. et al., Cardiovasc. Res. 40:389-395, 1998). Furthermore,
IL-6 concentrations increase over 48 hours from onset in the plasma
of patients with unstable angina with severe progression, but
decrease in those with uneventful progression (Biasucci, L. M. et
al., Circulation 99:2079-2084, 1999). This indicates that IL-6 may
be a useful indicator of disease progression. Plasma elevations of
IL-6 are associated with any nonspecific proinflammatory condition
such as trauma, infection, or other diseases that elicit an acute
phase response. IL-6 has a half-life of 4.2 hours in the
bloodstream and is elevated following acute myocardial infarction
and unstable angina (Manten, A. et al., Cardiovasc. Res.
40:389-395, 1998). The plasma concentration of IL-6 is elevated
within 8-12 hours of acute myocardial infarction onset, and can
approach 100 pg/ml. The plasma concentration of IL-6 in patients
with unstable angina was elevated at peak levels 72 hours after
onset, possibly due to the severity of insult (Biasucci, L. M. et
al., Circulation 94:874-877, 1996).
[0172] Interleukin-8 (IL-8) is a 6.5 kDa chemokine produced by
monocytes, endothelial cells, alveolar macrophages and fibroblasts.
IL-8 induces chemotaxis and activation of neutrophils and T
cells.
[0173] Tumor necrosis factor .alpha. (TNF.beta.) is a 17 kDa
secreted proinflammatory cytokine that is involved in the acute
phase response and is a pathogenic mediator of many diseases.
TNF.alpha. is normally produced by macrophages and natural killer
cells. TNF-alpha is a protein of 185 amino acids glycosylated at
positions 73 and 172. It is synthesized as a precursor protein of
212 amino acids. Monocytes express at least five different
molecular forms of TNF-alpha with molecular masses of 21.5-28 kDa.
They mainly differ by post-translational alterations such as
glycosylation and phosphorylation. The normal serum concentration
of TNF.alpha. is <40 pg/ml (2 pM). The plasma concentration of
TNF.alpha. is elevated in patients with acute myocardial
infarction, and is marginally elevated in patients with unstable
angina (Li, D. et al., Am. Heart J 137:1145-1152, 1999; Squadrito,
F. et al., Inflamm. Res. 45:14-19, 1996; Latini, R. et al., J.
Cardiovasc. Pharmacol. 23:1-6, 1994; Carlstedt, F. et al., J.
Intern. Med. 242:361-365, 1997). Elevations in the plasma
concentration of TNF.alpha. are associated with any proinflammatory
condition, including trauma, stroke, and infection. TNF.alpha. has
a half-life of approximately 1 hour in the bloodstream, indicating
that it may be removed from the circulation soon after symptom
onset. In patients with acute myocardial infarction, TNF.alpha. was
elevated 4 hours after the onset of chest pain, and gradually
declined to normal levels within 48 hours of onset (Li, D. et al.,
Am. Heart J. 137:1145-1152, 1999). The concentration of TNF.alpha.
in the plasma of acute myocardial infarction patients exceeded 300
pg/ml (15 pM) (Squadrito, F. et al., Inflamm. Res. 45:14-19, 1996).
Release of TNF.alpha. by monocytes has also been related to the
progression of pneumoconiosis in caol workers. Schins and Borm,
Occup. Environ. Med. 52: 441-50 (1995).
[0174] Soluble intercellular adhesion molecule (sICAM-1), also
called CD54, is a 85-110 kDa cell surface-bound immunoglobulin-like
integrin ligand that facilitates binding of leukocytes to
antigen-presenting cells and endothelial cells during leukocyte
recruitment and migration. sICAM-1 is normally produced by vascular
endothelium, hematopoietic stem cells and non-hematopoietic stem
cells, which can be found in intestine and epidermis. sICAM-1 can
be released from the cell surface during cell death or as a result
of proteolytic activity. The normal plasma concentration of sICAM-1
is approximately 250 ng/ml (2.9 nM). The plasma concentration of
sICAM-1 is significantly elevated in patients with acute myocardial
infarction and unstable angina, but not stable angina (Pellegatta,
F. et al., J. Cardiovasc. Pharmacol. 30:455-460, 1997; Miwa, K. et
al., Cardiovasc. Res. 36:37-44, 1997; Ghaisas, N. K. et al., Am. J.
Cardiol. 80:617-619, 1997; Ogawa, H. et al., Am. J. Cardiol.
83:38-42, 1999). Furthermore, ICAM-1 is expressed in
atherosclerotic lesions and in areas predisposed to lesion
formation, so it may be released into the bloodstream upon plaque
rupture (Iiyama, K. et al., Circ. Res. 85:199-207, 1999; Tenaglia,
A. N. et al., Am. J. Cardiol. 79:742-747, 1997). Elevations of the
plasma concentration of sICAM-1 are associated with ischemic
stroke, head trauma, atherosclerosis, cancer, preeclampsia,
multiple sclerosis, cystic fibrosis, and other nonspecific
inflammatory states (Kim, J. S., J. Neurol. Sci. 137:69-78, 1996;
Laskowitz, D. T. et al., J. Stroke Cerebrovasc. Dis. 7:234-241,
1998). The plasma concentration of sICAM-1 is elevated during the
acute stage of acute myocardial infarction and unstable angina. The
elevation of plasma sICAM-1 reaches its peak within 9-12 hours of
acute myocardial infarction onset, and returns to normal levels
within 24 hours (Pellegatta, F. et al., J. Cardiovasc. Pharmacol.
30:455-460, 1997). The plasma concentration of sICAM can approach
700 ng/ml (8 nM) in patients with acute myocardial infarction
(Pellegatta, F. et al., J. Cardiovasc. Pharmacol. 30:455-460,
1997). sICAM-1 is elevated in the plasma of individuals with acute
myocardial infarction and unstable angina, but it is not specific
for these diseases. It may, however, be useful marker in the
differentiation of acute myocardial infarction and unstable angina
from stable angina since plasma elevations are not associated with
stable angina. Interestingly, ICAM-1 is present in atherosclerotic
plaques, and may be released into the bloodstream upon plaque
rupture.
[0175] Vascular cell adhesion molecule (VCAM), also called CD106,
is a 100-110 kDa cell surface-bound immunoglobulin-like integrin
ligand that facilitates binding of B lymphocytes and developing T
lymphocytes to antigen-presenting cells during lymphocyte
recruitment. VCAM is normally produced by endothelial cells, which
line blood and lymph vessels, the heart, and other body cavities.
VCAM-1 can be released from the cell surface during cell death or
as a result of proteolytic activity. The normal serum concentration
of sVCAM is approximately 650 ng/ml (6.5 nM). The plasma
concentration of sVCAM-1 is marginally elevated in patients with
acute myocardial infarction, unstable angina, and stable angina
(Mulvihill, N. et al., Am. J. Cardiol. 83:1265-7, A9, 1999;
Ghaisas, N. K. et al., Am. J. Cardiol. 80:617-619, 1997). However,
sVCAM-1 is expressed in atherosclerotic lesions and its plasma
concentration may correlate with the extent of atherosclerosis
(Iiyama, K. et al., Circ. Res. 85:199-207, 1999; Peter, K. et al.,
Arterioscler. Thromb. Vasc. Biol. 17:505-512, 1997). Elevations in
the plasma concentration of sVCAM-1 are associated with ischemic
stroke, cancer, diabetes, preeclampsia, vascular injury, and other
nonspecific inflammatory states (Bitsch, A. et al., Stroke
29:2129-2135, 1998; Otsuki, M. et al., Diabetes 46:2096-2101, 1997;
Banks, R. E. et al., Br. J. Cancer 68:122-124, 1993; Steiner, M. et
al., Thromb. Haemost. 72:979-984, 1994; Austgulen, R. et al., Eur.
J. Obstet. Gynecol. Reprod. Biol. 71:53-58, 1997).
[0176] Monocyte chemotactic protein-1 (MCP-1) is a 10 kDa
chemotactic factor that attracts monocytes and basophils, but not
neutrophils or eosiniphils. MCP-1 is normally found in equilibrium
between a monomeric and homodimeric form, and it is normally
produced in and secreted by monocytes and vascular endothelial
cells (Yoshimura, T. et al., FEBS Lett. 244:487-493, 1989; Li, Y.
S. et al., Mol. Cell. Biochem. 126:61-68, 1993). MCP-1 has been
implicated in the pathogenesis of a variety of diseases that
involve monocyte infiltration, including psoriasis, rheumatoid
arthritis, and atherosclerosis. The normal concentration of MCP-1
in plasma is <0.1 ng/ml. The plasma concentration of MCP-1 is
elevated in patients with acute myocardial infarction, and may be
elevated in the plasma of patients with unstable angina, but no
elevations are associated with stable angina (Soejima, H. et al.,
J. Am. Coll. Cardiol. 34:983-988, 1999; Nishiyama, K. et al., Jpn.
Circ. J 62:710-712, 1998; Matsumori, A. et al., J. Mol. Cell.
Cardiol. 29:419-423, 1997). Interestingly, MCP-1 also may be
involved in the recruitment of monocytes into the arterial wall
during atherosclerosis. Elevations of the serum concentration of
MCP-1 are associated with various conditions associated with
inflammation, including alcoholic liver disease, interstitial lung
disease, sepsis, and systemic lupus erythematosus (Fisher, N. C. et
al., Gut 45:416-420, 1999; Suga, M. et al., Eur. Respir. J.
14:376-382, 1999; Bossink, A. W. et al., Blood 86:3841-3847, 1995;
Kaneko, H. et al. J. Rheumatol. 26:568-573, 1999). MCP-1 is
released into the bloodstream upon activation of monocytes and
endothelial cells. The concentration of MCP-1 in plasma form
patients with acute myocardial infarction has been reported to
approach 1 ng/ml (100 pM), and can remain elevated for one month
(Soejima, H. et al., J. Am. Coll. Cardiol. 34:983-988, 1999). MCP-1
is a specific marker of the presence of a pro-inflammatory
condition that involves monocyte migration.
[0177] Hemoglobin (Hb) is an oxygen-carrying iron-containing
globular protein found in erythrocytes. It is a heterodimer of two
globin subunits. .alpha..sub.2.gamma..sub.2 is referred to as fetal
Hb, .alpha..sub.2.beta..sub.2 is called adult HbA, and
.alpha..sub.2.delta..sub.2 is called adult HbA.sub.2. 90-95% of
hemoglobin is HbA, and the .alpha..sub.2 globin chain is found in
all Hb types, even sickle cell hemoglobin. Hb is responsible for
carrying oxygen to cells throughout the body. Hb.alpha..sub.2 is
not normally detected in serum.
[0178] Human lipocalin-type prostaglandin D synthase (hPDGS), also
called .beta.-trace, is a 30 kDa glycoprotein that catalyzes the
formation of prostaglandin D2 from prostaglandin H. The upper limit
of hPDGS concentrations in apparently healthy individuals is
reported to be approximately 420 ng/ml (Patent No. EP0999447A1).
Elevations of hPDGS have been identified in blood from patients
with unstable angina and cerebral infarction (Patent No.
EP0999447A1). Furthermore, hPDGS appears to be a useful marker of
ischemic episodes, and concentrations of hPDGS were found to
decrease over time in a patient with angina pectoris following
percutaneous transluminal coronary angioplasty (PTCA), suggesting
that the hPGDS concentration decreases as ischemia is resolved
(Patent No. EP0999447A1).
[0179] Mast cell tryptase, also known as alpha tryptase, is a 275
amino acid (30.7 kDa) protein that is the major neutral protease
present in mast cells. Mast cell tryptase is a specific marker for
mast cell activation, and is a marker of allergic airway
inflammation in asthma and in allergic reactions to a diverse set
of allergens. See, e.g., Taira et al., J. Asthma 39: 315-22 (2002);
Schwartz et al., N. Engl. J. Med. 316: 1622-26 (1987). Elevated
serum tryptase levels (>1 ng/mL) between 1 and 6 hours after an
event provides a specific indication of mast cell
degranulation.
[0180] Eosinophil cationic protein (ECP) is a heterogeneous protein
with molecular weight variants from 16-24 kDa and a pI of pH 10.8.
ECP is highly cytotoxic and is released by activated eosinophils.
Venge, Clinical and experimental allergy, 23 (suppl. 2): 3-7
(1993). Concentrations of ECP in the bronchoalveolar lavage fluid
(BALF) of asthma patients vary with the severity of their disease,
and ECP concentrations in sputum have also been shown to reflect
the pathophysiology of the disease. Bousquet et al., New Engl. J.
Med. 323: 1033-9 (1990). Virchow et al., Am. Rev. Respir. Dis. 146:
604-6 (1992). Assessment of serum ECP may be assumed to reflect
pulmonary inflammation in bronchial asthma. Koller et al., Arch.
Dis. Childhood 73: 413-7 (1995); see also, Sorkness et al., Clin.
Exp. Allergy 32: 1355-59 (2002); Badr-elDin et al., East Mediterr.
Health J. 5: 664-75 (1999).
[0181] KL-6 (also referred to as MUC1) is a high molecular weight
(>300 kDa) mucinous glycoprotein expressed on pneumonocytes.
Serum levels of KL-6 are reportedly elevated in interstitial lung
diseases, which are characterized by exertional dyspnea. KL-6 has
been shown to be a marker of various interstitial lung diseases,
including pulmonary fibrosis, interstitial pneumonia, sarcoidosis,
and interstitial pneumonitis. See, e.g., Kobayashi and Kitamura,
Chest 108: 311-15 (1995); Kohno, J. Med. Invest. 46: 151-58 (1999);
Bandoh et al., Ann. Rheum. Dis. 59: 257-62 (2000); and Yamane et
al., J. Rheumatol. 27: 930-4 (2000).
[0182] Procalcitonin is a 116 amino acid (14.5 kDa) protein encoded
by the Calc-1 gene located on chromosome 11p15.4. The Calc-1 gene
produces two transcripts that are the result of alternative
splicing events. Pre-procalcitonin contains a 25 amino acid signal
peptide which is processed by C-cells in the thyrois to a 57 amino
acid N-terminal fragment, a 32 amino acid calcitonin fragment, and
a 21 amino acid katacalcin fragment. Procalcitonin is secreted
intact as a glycosylated product by other body cells. Whicher et
al., Ann. Clin. Biochem. 38: 483-93 (2001). Plasma procalcitonin
has been identified as a marker of sepsis and its severity (Yukioka
et al., Ann. Acad. Med. Singapore 30: 528-31 (2001)), with day 2
procalcitonin levels predictive of mortality (Pettila et al.,
Intensive Care Med. 28: 1220-25 (2002).
[0183] Interleukin 10 ("IL-0") is a 160 amino acid (18.5 kDa
predicted mass) cytokine that is a member of the four .alpha.-helix
bundle family of cytokines. In solution, IL-10 forms a homodimer
having an apparent molecular weight of 39 kDa. The human IL-10 gene
is located on chromosome 1. Viera et al., Proc. Natl. Acad. Sci.
USA 88: 1172-76 (1991); Kim et al., J. Immunol. 148: 3618-23
(1992). Overproduction of IL-10 has been identified as a marker in
sepsis, and is predictive of severity and mortality. Gogos et al.,
J. Infect. Dis. 181: 176-80 (2000).
[0184] (v) Exemplary Specific Markers for Neural Tissue Injury
[0185] Adenylate kinase (AK) is a ubiquitous 22 kDa cytosolic
enzyme that catalyzes the interconversion of ATP and AMP to ADP.
Four isoforms of adenylate kinase have been identified in mammalian
tissues (Yoneda, T. et al., Brain Res Mol Brain Res 62:187-195,
1998). The AK1 isoform is found in brain, skeletal muscle, heart,
and aorta. The normal serum mass concentration of AK1 is currently
unknown, because a functional assay is typically used to measure
total AK concentration. The normal serum AK concentration is <5
units/liter and AK elevations have been performed using CSF
(Bollensen, E. et al., Acta Neurol Scand 79:53-582, 1989). Serum
AK1 appears to have the greatest specificity of the AK isoforms as
a marker of cerebral injury. AK may be best suited as a
cerebrospinal fluid marker of cerebral ischemia, where its dominant
source would be neural tissue.
[0186] Neurotrophins are a family of growth factors expressed in
the mammalian nervous system. Some examples include nerve growth
factor (NGF), brain-derived neurotrophic factor (BDNF),
neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-4/5). Neurotrophins
exert their effects primarily as target-derived paracrine or
autocrine neurotrophic factors. The role of the neurotrophins in
survival, differentiation and maintenance of neurons is well known.
They exhibit partially overlapping but distinct patterns of
expression and cellular targets. In addition to the effects in the
central nervous system, neurotrophins also affect peripheral
afferent and efferent neurons.
[0187] BDNF is a potent neurotrophic factor which supports the
growth and survivability of nerve and/or glial cells. BDNF is
expressed as a 32 kDa precursor "pro-BDNF" molecule that is cleaved
to a mature BDNF form. Mowla et al., J. Biol. Chem. 276: 12660-6
(2001). The most abundant active form of human BDNF is a 27 kDa
homodimer, formed by two identical 119 amino acid subunits, which
is held together by strong hydrophobic interactions; however,
pro-BDNF is also released extracellularly and is biologically
active. BDNF is widely distributed throughout the CNS and displays
in vitro trophic effects on a wide range of neuronal cells,
including hippocampal, cerebellar, and cortical neurons. In vivo,
BDNF has been found to rescue neural cells from traumatic and toxic
brain injury. For example, studies have shown that after transient
middle cerebral artery occlusion, BDNF mRNA is upregulated in
cortical neurons (Schabiltz et al., J. Cereb. Blood Flow Metab.
14:500-506, 1997). In experimentally induced focal, unilateral
thrombotic stroke, BDNF mRNA was increased from 2 to 18 h following
the stroke. Such results suggest that BDNF potentially plays a
neuroprotective role in focal cerebral ischemia.
[0188] NT-3 is also a 27 kDa homodimer consisting of two 119-amino
acid subunits. The addition of NT-3 to primary cortical cell
cultures has been shown to exacerbate neuronal death caused by
oxygen-glucose deprivation, possible via oxygen free radical
mechanisms (Bates et al., Neurobiol. Dis. 9:24-37, 2002). NT-3 is
expressed as an inactive pro-NT-3 molecule, which is cleaved to the
mature biologically active form.
[0189] Calbindin-D is a 28 kDa cytosolic vitamin D-dependent
Ca.sup.2+-binding protein that may serve a cellular protective
function by stabilizing intracellular calcium levels. Calbindin-D
is found in the central nervous system, mainly in glial cells, and
in cells of the distal renal tubule (Hasegawa, S. et al., J. Urol.
149:1414-1418, 1993). The normal serum concentration of calbindin-D
is <20 pg/ml (0.7 pM). Serum calbindin-D concentration is
reportedly elevated following cardiac arrest, and this elevation is
thought to be a result of CNS damage due to cerebral ischemia
(Usui, A. et al., J. Neurol. Sci. 123:134-139, 1994). Elevations of
serum calbindin-D are elevated and plateau soon after reperfusion
following ischemia. Maximum serum calbindin-D concentrations can be
as much as 700 pg/ml (25 pM).
[0190] Creatine kinase (CK) is a cytosolic enzyme that catalyzes
the reversible formation of ADP and phosphocreatine from ATP and
creatine. The brain-specific CK isoform (CK-BB) is an 85 kDa
cytosolic protein that accounts for approximately 95% of the total
brain CK activity. It is also present in significant quantities in
cardiac tissue, intestine, prostate, rectum, stomach, smooth
muscle, thyroid uterus, urinary bladder, and veins (Johnsson, P.
J., Cardiothorac. Vasc. Anesth. 10: 120-126, 1996). The normal
serum concentration of CK-BB is <10 ng/ml (120 pM). Serum CK-BB
is elevated after hypoxic and ischemic brain injury, but a further
investigation is needed to identify serum elevations in specific
stroke types (Laskowitz, D. T. et al., J. Stroke Cerebrovasc. Dis.
7:234-241, 1998). Elevations of CK-BB in serum can be attributed to
cerebral injury due to ischemia, coupled with increased
permeability of the blood brain barrier. No correlation of the
serum concentration of CK-BB with the extent of damage (infarct
volume) or neurological outcome has been established. CK-BB has a
half-life of 1-5 hours in serum and is normally detected in serum
at a concentration of <10 ng/ml (120 pM). In severe stroke,
serum concentrations CK-BB are elevated and peak soon after the
onset of stroke (within 24 hours), gradually returning to normal
after 3-7 days (4). CK-BB concentrations in the serum of
individuals with head injury peak soon after injury and return to
normal between 3.5-12 hours after injury, depending on the injury
severity (Skogseid, I. M. et al., Acta Neurochir. (Wien.)
115:106-111, 1992). Maximum serum CK-BB concentrations can exceed
250 ng/ml (3 rM). CK-BB may be best suited as a CSF marker of
cerebral ischemia, where its dominant source would be neural
tissue. CKBB might be more suitable as a serum marker of CNS damage
after head injury because it is elevated for a short time in these
individuals, with its removal apparently dependent upon the
severity of damage.
[0191] Glial fibrillary acidic protein (GFAP) is a 55 kDa cytosolic
protein that is a major structural component of astroglial
filaments and is the major intermediate filament protein in
astrocytes. GFAP is specific to astrocytes, which are interstitial
cells located in the CNS and can be found near the blood-brain
barrier. GFAP is not normally detected in serum. Serum GFAP is
elevated following ischemic stroke (Niebroj-Dobosz, I., et al.,
Folia Neuropathol. 32:129-137, 1994). Current reports investigating
serum GFAP elevations associated with stroke are severely limited,
and much further investigation is needed to establish GFAP as a
serum marker for all stroke types. Most studies investigating GFAP
as a stroke marker have been performed using cerebrospinal fluid.
Elevations of GFAP in serum can be attributed to cerebral injury
due to ischemia, coupled with increased permeability of the blood
brain barrier. No correlation of the serum concentration of GFAP
with the extent of damage (infarct volume) or neurological outcome
has been established. GFAP is elevated in cerebrospinal fluid of
individuals with various neuropathies affecting the CNS, but there
are no reports currently available describing the release of GFAP
into the serum of individuals with diseases other than stroke
(Albrechtsen, M. and Bock, E. J., Neuroimmunol. 8:301-309, 1985).
Serum concentrations GFAP appear to be elevated soon after the
onset of stroke, continuously increase and persist for an amount of
time (weeks) that may correlate with the severity of damage. GFAP
appears to a very specific marker for severe CNS injury,
specifically, injury to astrocytes due to cell death caused by
ischemia or physical damage.
[0192] Lactate dehydrogenase (LDH) is a ubiquitous 135 kDa
cytosolic enzyme. It is a tetramer of A and B chains that catalyzes
the reduction of pyruvate by NADH to lactate. Five isoforms of LDH
have been identified in mammalian tissues, and the tissue-specific
isoforms are made of different combinations of A and B chains. The
normal serum mass concentration of LDH is currently unknown,
because a functional assay is typically used to measure total LDH
concentration. The normal serum LDH concentration is <600
units/liter (Ray, P. et al., Cancer Detect. Prev. 22:293-304,
1998). A great majority of investigations into LDH elevations in
the context of stroke have been performed using cerebrospinal
fluid, and elevations correlate with the severity of injury.
Elevations in serum LDH activity are reported following both
ischemic and hemorrhagic stroke, but further studies are needed in
serum to confirm this observation and to determine a correlation
with the severity of injury and neurological outcome (Aggarwal, S.
P. et al., J. Indian Med. Assoc. 93:331-332, 1995; Maiuri, F. et
al., Neurol. Res. 11:6-8, 1989). LDH may be best suited as a
cerebrospinal fluid marker of cerebral ischemia, where its dominant
source would be neural tissue.
[0193] Myelin basic protein (MBP) is actually a 14-21 kDa family of
cytosolic proteins generated by alternative splicing of a single
MBP gene that is likely involved in myelin compaction around axons
during the myelination process. MBP is specific to oligodendrocytes
in the CNS and in Schwann cells of the peripheral nervous system
(PNS). It accounts for approximately 30% of the total myelin
protein in the CNS and approximately 10% of the total myelin
protein in the PNS. The normal serum concentration of MBP is <7
ng/ml (400 pM). Serum MBP is elevated after all types of severe
stroke, specifically thrombotic stroke, embolic stroke,
intracerebral hemorrhage, and subarachnoid hemorrhage, while
elevations in MBP concentration are not reported in the serum of
individuals with strokes of minor to moderate severity, which would
include lacunar infarcts or transient ischemic attacks (Palfreyman,
J. W. et al., Clin. Chim. Acta 92:403-409, 1979). Elevations of MBP
in serum can be attributed to cerebral injury due to physical
damage or ischemia caused by infarction or cerebral hemorrhage,
coupled with increased permeability of the blood brain barrier. The
serum concentration of MBP has been reported to correlate with the
extent of damage (infarct volume), and it may also correlate with
neurological outcome. The amount of available information regarding
serum MBP elevations associated with stroke is limited, because
most investigations have been performed using cerebrospinal fluid.
MBP is normally detected in serum at an upper limit of 7 ng/ml (400
pM), is elevated after severe stroke and cerebral injury. Serum MBP
is thought to be elevated within hours after stroke onset, with
concentrations increasing to a maximum level within 2-5 days after
onset. After the serum concentration reaches its maximum, which can
exceed 120 ng/ml (6.9 nM), it can take over one week to gradually
decrease to normal concentrations. Because the severity of damage
has a direct effect on the release of MBP, it will affect the
release kinetics by influencing the length of time that MBP is
elevated in the serum. MBP will be present in the serum for a
longer period of time as the severity of injury increases. The
release of MBP into the serum of patients with head injury is
thought to follow similar kinetics as those described for stroke,
except that serum MBP concentrations reportedly correlate with the
neurological outcome of individuals with head injury (Thomas, D. G.
et al., Acta Neurochir. Suppl. (Wien) 28:93-95, 1979). The release
of MBP into the serum of patients with intracranial tumors is
thought to be persistent, but still needs investigation. Finally,
serum MBP concentrations can sometimes be elevated in individuals
with demyelinating diseases, but no conclusive investigations have
been reported. As reported in individuals with multiple sclerosis,
MBP is frequently elevated in the cerebrospinal fluid, but matched
elevations in serum are often not present (Jacque, C. et al., Arch.
Neurol. 39:557-560, 1982). This could indicate that cerebral damage
has to be accompanied by an increase in the permeability of the
blood-brain barrier to result in elevation of serum MBP
concentrations. However, MBP can also be elevated in the population
of individuals having intracranial tumors. The presence of these
individuals in the larger population of individuals that would be
candidates for an assay using this marker for stroke is rare. These
individuals, in combination with individuals undergoing
neurosurgical procedures or with demyelinating diseases, would
nonetheless have an impact on determining the specificity of MBP
for cerebral injury. Additionally, serum MBP may be useful as a
marker of severe stroke, potentially identifying individuals that
would not benefit from stroke therapies and treatments, such as tPA
administration.
[0194] Neural cell adhesion molecule (NCAM), also called CD56, is a
170 kDa cell surface-bound immunoglobulin-like integrin ligand that
is involved in the maintenance of neuronal and glial cell
interactions in the nervous system, where it is expressed on the
surface of astrocytes, oligodendrocytes, Schwann cells, neurons,
and axons. NCAM is also localized to developing skeletal muscle
myotubes, and its expression is upregulated in skeletal muscle
during development, denervation and renervation. The normal serum
mass concentration of NCAM has not been reported. NCAM is commonly
measured by a functional enzyme immunoassay and is reported to have
a normal serum concentration of <20 units/ml. Changes in serum
NCAM concentrations specifically related to stroke have not been
reported. NCAM may be best suited as a CSF marker of cerebral
ischemia, where its dominant source would be neural tissue.
[0195] Enolase is a 78 kDa homo- or heterodimeric cytosolic protein
produced from .alpha., .beta., and .gamma. subunits. It catalyzes
the interconversion of 2-phosphoglycerate and phosphoenolpyruvate
in the glycolytic pathway. Enolase can be present as
.alpha..alpha., .beta..beta., .alpha..gamma., and .gamma..gamma.
isoforms. The .alpha. subunit is found in glial cells and most
other tissues, the .beta. subunit is found in muscle tissue, and
the .gamma. subunit if found mainly in neuron al and neuroendocrine
cells (Quinn, G. B. et al., Clin. Chem. 40:790-795, 1994). The
.gamma..gamma. enolase isoform is most specific for neurons, and is
referred to as neuron-specific enolase (NSE). NSE, found
predominantly in neurons and neuroendocrine cells, is also present
in platelets and erythrocytes. The normal serum concentration of
NSE is <12.5 ng/ml (160 pM). NSE is made up of two subunits;
thus, the most feasible immunological assay used to detect NSE
concentrations would be one that is directed against one of the
subunits. In this case, the .gamma. subunit would be the ideal
choice. However, the .gamma. subunit alone is not as specific for
cerebral tissue as the .gamma. isoform, since a measurement of the
.gamma. subunit alone would detect both the .alpha..gamma. and
.gamma..gamma. isoforms. In this regard, the best immunoassay for
NSE would be a two-site assay that could specifically detect the
.gamma..gamma. isoform. Serum NSE is reportedly elevated after all
stroke types, including TIAs, which are cerebral in origin and are
thought to predispose an individual to having a more severe stroke
at a later date (Isgro, F. et al., Eur. J. Cardiothorac. Surg.
11:640-644, 1997). Elevations of NSE in serum can be attributed to
cerebral injury due to physical damage or ischemia caused by
infarction or cerebral hemorrhage, coupled with increased
permeability of the blood brain barrier, and the serum
concentration of NSE has been reported to correlate with the extent
of damage (infarct volume) and neurological outcome (Martens, P. et
al., Stroke 29:2363-2366, 1998). Additionally, a secondary
elevation of serum NSE concentration may be an indicator of delayed
neuronal injury resulting from cerebral vasospasm (Laskowitz, D. T.
et al., J. Stroke Cerebrovasc. Dis. 7, 234-241, 1998). NSE, which
has a biological half-life of 48 hours and is normally detected in
serum at an upper limit of 12.5 ng/ml (160 pM), is elevated after
stroke and cerebral injury. Serum NSE is elevated after 4 hours
from stroke onset, with concentrations reaching a maximum 1-3 days
after onset (Missler, U. et al., Stroke 28:1956-1960, 1997). After
the serum concentration reaches its maximum, which can exceed 300
ng/ml (3.9 nM), it gradually decreases to normal concentrations
over approximately one week. Because the severity of damage has a
direct effect on the release of NSE, it will affect the release
kinetics by influencing the length of time that NSE is elevated in
the serum. NSE will be present in the serum for a longer period of
time as the severity of injury increases. The release of NSE into
the serum of patients with head injury follows different kinetics
as seen with stroke, with the maximum serum concentration being
reached within 1-6 hours after injury, often returning to baseline
within 24 hours (Skogseid, I. M. et al., Acta Neurochir. (Wien.)
115:106-111, 1992). NSE is a specific marker for cerebral injury,
specifically, injury to neuronal cells due to cell death caused by
ischemia or physical damage. Neurons are about 10-fold less
abundant in the brain than glial cells, so any cerebral injury
coupled with increased permeability of the blood-brain barrier will
have to occur in a region that has a significant regional
population of neurons to significantly increase the serum NSE
concentration. In addition, elevated serum concentrations of NSE
can also indicate complications related to cerebral injury after
AMI and cardiac surgery. Elevations in the serum concentration of
NSE correlate with the severity of damage and the neurological
outcome of the individual. NSE can be used as a marker of all
stroke types, including TIAs. However, NSE cannot be used to
differentiate ischemic and hemorrhagic stroke, and it is elevated
in the population of individuals having tumors with neuroendocrine
features.
[0196] Proteolipid protein (PLP) is a 30 kDa integral membrane
protein that is a major structural component of CNS myelin. PLP is
specific to oligodendrocytes in the CNS and accounts for
approximately 50% of the total CNS myelin protein in the central
sheath, although extremely low levels of PLP have been found
(<1%) in peripheral nervous system (PNS) myelin. The normal
serum concentration of PLP is <9 ng/ml (300 pM). Serum PLP is
elevated after cerebral infarction, but not after transient
ischemic attack (Trotter, J. L. et al., Ann. Neurol. 14:554-558,
1983). Current reports investigating serum PLP elevations
associated with stroke are severely limited. Elevations of PLP in
serum can be attributed to cerebral injury due to physical damage
or ischemia caused by infarction or cerebral hemorrhage, coupled
with increased permeability of the blood brain barrier. Correlation
of the serum concentration of PLP with the extent of damage
(infarct volume) or neurological outcome has not been established.
No investigations examining the release kinetics of PLP into serum
and its subsequent removal have been reported, but maximum
concentrations approaching 60 ng/ml (2 nM) have been reported in
encephalitis patients, which nearly doubles the concentrations
found following stroke. PLP appears to a very specific marker for
severe CNS injury, specifically, injury to oligodendrocytes. The
available information relating PLP serum elevations and stroke is
severely limited. PLP is also elevated in the serum of individuals
with various neuropathies affecting the CNS. The undiagnosed
presence of these individuals in the larger population of
individuals that would be candidates for an assay using this marker
for stroke is rare.
[0197] S-100 is a 21 kDa homo- or heterodimeric cytosolic
Ca.sup.2+-binding protein produced from .alpha. and .beta.
subunits. It is thought to participate in the activation of
cellular processes along the Ca2+-dependent signal transduction
pathway (Bonfrer, J. M. et al., Br. J. Cancer 77:2210-2214, 1998).
S-100ao (.alpha..alpha. isoform) is found in striated muscles,
heart and kidney, S-100a (.alpha..beta. isoform) is found in glial
cells, but not in Schwann cells, and S-100b (.beta..beta. isoform)
is found in high concentrations in glial cells and Schwann cells,
where it is a major cytosolic component. The .beta. subunit is
specific to the nervous system, predominantly the CNS, under normal
physiological conditions and, in fact, accounts for approximately
96% of the total S-100 protein found in the brain (Jensen, R. et
al., J. Neurochem. 45:700-705, 1985). In addition, S-100.beta. can
be found in tumors of neuroendocrine origin, such as gliomas,
melanomas, Schwannomas, neurofibromas, and highly differentiated
neuroblastomas, like ganglioneuroblastoma and ganglioneuroma
(Persson, L. et al., Stroke 18:911-918, 1987). The normal serum
concentration of S-100.beta. is <0.2 ng/ml (19 pM), which is the
detection limit of the immunological detection assays used. Serum
S-100.beta. is elevated after all stroke types, including TIAs.
Elevations of S-100.beta. in serum can be attributed to cerebral
injury due to physical damage or ischemia caused by infarction or
cerebral hemorrhage, coupled with increased permeability of the
blood-brain barrier, and the serum concentration of S-100b has been
shown to correlate with the extent of damage (infarct volume) and
neurological outcome (Martens, P. et al., Stroke 29:2363-2366,
1998; Missler, U. et al., Stroke 28:1956-1960, 1997). S-100b has a
biological half-life of 2 hours and is not normally detected in
serum, but is elevated after stroke and cerebral injury. Serum
S-100.beta. is elevated after 4 hours from stroke onset, with
concentrations reaching a maximum 2-3 days after onset. After the
serum concentration reaches its maximum, which can approach 20
ng/ml (1.9 mM), it gradually decreases to normal over approximately
one week. Because the severity of damage has a direct effect on the
release of S-100b, it will affect the release kinetics by
influencing the length of time that S-100b is elevated in the
serum. S-100b will be present in the serum for a longer period of
time as the seventy of injury increases. The release of S-100b into
the serum of patients with head injury seems to follow somewhat
similar kinetics as reported with stroke, with the only exception
being that serum S-100.beta. can be detected within 2.5 hours of
onset and the maximum serum concentration is reached approximately
1 day after onset (Woertgen, C. et al., Acta Neurochir. (Wien)
139:1161-1164, 1997). S-100.beta. is a specific marker for cerebral
injury, specifically, injury to glial cells due to cell death
caused by ischemia or physical damage. Glial cells are about 10
times more abundant in the brain than neurons, so any cerebral
injury coupled with increased permeability of the blood-brain
barrier will likely produce elevations of serum S-100.beta..
Furthermore, elevated serum concentrations of S-100b can indicate
complications related to cerebral injury after AMI and cardiac
surgery. S-100b has been virtually undetectable in normal
individuals, and elevations in its serum concentration correlate
with the seventy of damage and the neurological outcome of the
individual. S-100b can be used as a marker of all stroke types,
including TIAs. However, S-100b cannot be used to differentiate
ischemic and hemorrhagic stroke, and it is elevated in the
population of individuals having neuroendocrine tumors, usually in
advanced stages.
[0198] Thrombomodulin (TM) is a 70 kDa single chain integral
membrane glycoprotein found on the surface of vascular endothelial
cells. TM demonstrates anticoagulant activity by changing the
substrate specificity of thrombin. The formation of a 1:1
stoichiometric complex between thrombin and TM changes thrombin
function from procoagulant to anticoagulant. This change is
facilitated by a change in thrombin substrate specificity that
causes thrombin to activate protein C (an inactivator of factor Va
and factor VIIIa), but not cleave fibrinogen or activate other
coagulation factors (Davie, E. W. et al., Biochem. 30:10363-10370,
1991). The normal serum concentration of TM is 25-60 ng/ml (350-850
pM). Current reports describing serum TM concentration alterations
following ischemic stroke are mixed, reporting no changes or
significant increases (Seki, Y. et al., Blood Coagul. Fibrinolysis
8:391-396, 1997). Serum elevations of TM concentration reflect
endothelial cell injury and would not indicate coagulation or
fibrinolysis activation.
[0199] The gamma isoform of protein kinase C (PKCg) is specific for
CNS tissue and is not normally found in the circulation. PKCg is
activated during cerebral ischemia and is present in the ischemic
penumbra at levels 2-24-fold higher than in contralateral tissue,
but is not elevated in infarcted tissue (Krupinski, J. et al., Acta
Neurobiol. Exp. (Warz) 58:13-21, 1998). In addition, animal models
have identified increased levels of PKCg in the peripheral
circulation of rats following middle cerebral artery occlusion
(Cornell-Bell, A. et al., Patent No. WO 01/16599 A1). Additional
isoforms of PKC, beta I and beta II were found in increased levels
in the infarcted core of brain tissue from patients with cerebral
ischemia (Krupinski, J. et al., Acta Neurobiol. Exp. (Warz)
58:13-21, 1998). Furthermore, the alpha and delta isoforms of PKC
(PKCa and PKCd, respectively) have been implicated in the
development of vasospasm following subarachnoid hemorrhage using a
canine model of hemorrhage. PKCd expression was significantly
elevated in the basilar artery during the early stages of
vasospasm, and PKCa was significantly elevated as vasospasm
progressed (Nishizawa, S. et al., Eur. J. Pharmacol. 398:113-119,
2000). Therefore, it may be of benefit to measure various isoforms
of PKC, either individually or in various combinations thereof, for
the identification of cerebral damage, the presence of the ischemic
penumbra, as well as the development and progression of cerebral
vasospasm following subarachnoid hemorrhage. Ratios of PKC isoforms
such as PKCg and either PKCbI, PKCbII, or both also may be of
benefit in identifying a progressing stroke, where the ischemic
penumbra is converted to irreversibly damaged infarcted tissue. In
this regard, PKCg may be used to identify the presence and volume
of the ischemic penumbra, and either PKCbI, PKCbII, or both may be
used to identify the presence and volume of the infarcted core of
irreversibly damaged tissue during stroke. PKCd, PKCa, and ratios
of PKCd and PKCa may be useful in identifying the presence and
progression of cerebral vasospasm following subarachnoid
hemorrhage.
[0200] (vi) Exemplary Non-Specific Markers Related to Coagulation
and Hemostasis
[0201] Plasmin is a 78 kDa serine proteinase that proteolytically
digests crosslinked fibrin, resulting in clot dissolution. The 70
kDa serine proteinase inhibitor .alpha.2-antiplasmin (.alpha.2AP)
regulates plasmin activity by forming a covalent 1:1 stoichiometric
complex with plasmin. The resulting .about.150 kDa
plasmin-.alpha.2AP complex (PAP), also called plasmin inhibitory
complex (PIC) is formed immediately after .alpha.2AP comes in
contact with plasmin that is activated during fibrinolysis. The
normal serum concentration of PAP is <1 .mu.g/ml (6.9 nM). Serum
PAP concentration is significantly elevated following embolic and
hemorrhagic stroke, but not thrombotic or lacunar stroke, and the
magnitude of elevation correlates with the severity of injury and
neurological outcome (Seki, Y. et al., Am. J. Hematol. 50:155-160,
1995; Yamazaki, M. et al., Blood Coagul. Fibrinolysis 4:707-712,
1993; Uchiyama, S. et al., Semin. Thromb. Hemost. 23:535-541, 1997;
Fujii, Y. et al., Neurosurgery 37:226-234, 1995). There are no
reports that identify elevations in serum PAP concentration
following cerebral transient ischemic attacks. Elevations in the
serum concentration of PAP can be attributed to the activation of
fibrinolysis. Elevations in the serum concentration of PAP may be
associated with clot presence, or any condition that causes or is a
result of fibrinolysis activation. These conditions can include
atherosclerosis, disseminated intravascular coagulation, AMI,
surgery, trauma, unstable angina, and thrombotic thrombocytopenic
purpura. PAP is formed immediately following proteolytic activation
of plasmin. Serum PAP is increased in embolic and hemorrhagic
stroke. Serum concentrations are elevated soon after stroke onset
and may persist for over 2 weeks (Fujii, Y. et al., J. Neurosurg.
86:594-602, 1997). In addition, serum PAP concentration may be
higher in hemorrhagic stroke than in ischemic stroke. This could
reflect the increased magnitude of coagulation activation
associated with hemorrhage. Serum concentrations of PAP associated
with stroke can approach 0.6 .mu.g/ml (41 nM). PAP is a specific
marker for fibrinolysis activation and the presence of a recent or
continual hypercoagulable state. It is not specific for stroke or
cerebral injury and can be elevated in many other disease states.
However, it may be possible to use PAP to differentiate hemorrhagic
stroke from ischemic stroke, which would be beneficial in ruling
out patients for thrombolytic therapy, and to identify embolic vs.
non-embolic ischemic strokes.
[0202] .beta.-thromboglobulin (PTG) is a 36 kDa platelet a granule
component that is released upon platelet activation. The normal
serum concentration of .beta.TG is <40 ng/ml (1.1 nM). Serum
.beta.TG concentration is elevated following ischemic and
hemorrhagic stroke (Landi, G. et al., Neurol. 37:1667-1671, 1987).
Serum elevations were not found to correlate with injury severity
or neurological outcome. Investigations regarding .beta.TG serum
elevations in stroke are severely limited. Elevations in the serum
PTG concentration can be attributed to platelet activation, which
could indirectly indicate the presence of vascular injury.
Elevations in the serum concentration of .beta.TG may be associated
with clot presence, or any condition that causes platelet
activation. These conditions can include atherosclerosis,
disseminated intravascular coagulation, AMI, surgery, trauma,
unstable angina, and thrombotic thrombocytopenic purpura. .beta.TG
is released into the circulation immediately after platelet
activation and aggregation. It has a biphasic half-life of 10
minutes, followed by an extended 1 hour half-life in serum
(Switaiska, H. I. et al., J. Lab. Clin. Med. 106:690-700, 1985).
Serum .beta.TG concentration is reported to be elevated in various
stroke types, but these studies may not be completely reliable.
Special precautions must be taken to avoid platelet activation
during the blood sampling process. Platelet activation is common
during regular blood sampling, and could lead to artificial
elevations of serum .beta.TG concentration. In addition, the amount
of .beta.TG released into the bloodstream is dependent on the
platelet count of the individual, which can be quite variable.
Serum concentrations of PTG associated with stroke can approach 70
ng/ml (2 nM). .beta.TG is a specific marker of platelet activation,
but it is not specific for stroke or cerebral injury and can be
elevated in many other disease states.
[0203] Platelet factor 4 (PF4) is a 40 kDa platelet a granule
component that is released upon platelet activation. PF4 is a
marker of platelet activation and has the ability to bind and
neutralize heparin. The normal serum concentration of PF4 is <7
ng/ml (175 pM). Serum PF4 concentration is marginally elevated
following intracerebral infarction, but not in individuals with
intracerebral hemorrhage (Carter, A. M. et al., Arterioscler.
Thromb. Vasc. Biol. 18:1124-1131, 1998). Additionally, serum PF4
concentrations are increased 5-9 days following subarachnoid
hemorrhage, which may be related to the onset of cerebral vasospasm
(Hirashima, Y. et al., Neurochem. Res. 22:1249-1255, 1997).
Investigations regarding PF4 serum elevations in stroke are
severely limited. Elevations in the serum PF4 concentration can be
attributed to platelet activation, which could indirectly indicate
the presence of vascular injury. Elevations in the serum
concentration of PF4 may be associated with clot presence, or any
condition that causes platelet activation. These conditions can
include atherosclerosis, disseminated intravascular coagulation,
AMI, surgery, trauma, unstable angina, and thrombotic
thrombocytopenic purpura. PF4 is released into the circulation
immediately after platelet activation and aggregation. It has a
biphasic half-life of 1 minute, followed by an extended 20 minute
half-life in serum. The half-life of PF4 in serum can be extended
to 20-40 minutes by the presence of heparin (Rucinski, B. et al.,
Am. J. Physiol. 251:H800-H807, 1986). Special precautions must be
taken to avoid platelet activation during the blood sampling
process. Serum concentrations of PF4 associated with stroke can
exceed 200 ng/ml (5 nM), but it is likely that this value may be
influenced by platelet activation during the sampling procedure.
Furthermore, the serum PF4 concentration would be dependent on
platelet count, requiring a second variable to be determined along
with the concentration estimates. Finally, patients taking aspirin
or other platelet activation inhibitors would compromise the
clinical usefulness of PF4 as a marker of platelet activation.
[0204] Fibrinopeptide A (FPA) is a 16 amino acid, 1.5 kDa peptide
that is liberated from amino terminus of fibrinogen by the action
of thrombin. Fibrinogen is synthesized and secreted by the liver.
The normal serum concentration of FPA is <4 ng/ml (2.7 nM).
Serum FPA is elevated after all stroke types, including cerebral
transient ischemic attacks (TIAs) (Fon, E. A. et al., Stroke
25:282-286, 1994; Tohgi, H. et al., Stroke 21:1663-1667, 1990;
Landi, G. et al., Neurol. 37:1667-1671, 1987). Elevations of FPA in
serum can be attributed to coagulation activation, and the serum
concentration of FPA has been reported to correlate with the
neurological outcome, but not the severity or extent of damage
(infarct volume) (Feinberg, W. M. et al., Stroke 27:1296-1300,
1996). Elevations in the serum concentration of FPA are associated
with any condition that causes or is a result of coagulation
activation. These conditions can include AMI, surgery, cancer,
disseminated intravascular coagulation, nephrosis, thrombotic
thrombocytopenic purpura, and unstable angina. FPA is released into
the bloodstream immediately upon clot formation and it can remain
elevated for more than 1 month. Maximum serum FPA concentrations
following stroke can exceed 50 ng/ml (34 nM).
[0205] (vii) Other Non-Specific Markers for Cellular Injury
[0206] Human vascular endothelial growth factor (VEGF) is a dimeric
protein, the reported activities of which include stimulation of
endothelial cell growth, angiogenesis, and capillary permeability.
VEGF is secreted by a variety of vascularized tissues. In an
oxygen-deficient environment, vascular endothelial cells may be
damaged and may not ultimately survive. However, such endothelial
damage stimulates VEGF production by vascular smooth muscle cells.
Vascular endothelial cells may exhibit increased survival in the
presence of VEGF, an effect that is believed to be mediated by
expression of Bcl-2. VEGF can exist as a variety of splice variants
known as VEGF(189), VEGF(165), VEGF(164), VEGFB(155), VEGF(148),
VEGF(145), and VEGF(121).
[0207] Insulin-like growth factor-1 (IGF-1) is a ubiquitous 7.5 kDa
secreted protein that mediates the anabolic and somatogenic effects
of growth hormone during development (1, 2). In the circulation,
IGF-1 is normally bound to an IGF-binding protein that regulates
IGF activity. The normal serum concentration of IGF-1 is
approximately 160 ng/ml (21.3 nM). Serum IGF-1 concentrations are
reported to be significantly decreased in individuals with ischemic
stroke, and the magnitude of reduction appears to correlate with
the severity of injury (Schwab, S. et al., Stroke 28:1744-1748,
1997). Decreased IGF-1 serum concentrations have been reported in
individuals with trauma and massive activation of the immune
system. Due to its ubiquitous expression, serum IGF-1
concentrations could also be decreased in cases of non-cerebral
ischemia. Interestingly, IGF-1 serum concentrations are decreased
following ischemic stroke, even though its cellular expression is
upregulated in the infarct zone (Lee, W. H. and Bondy, C., Ann.
N.Y. Acad. Sci. 679:418-422, 1993). The decrease in serum
concentration could reflect an increased demand for growth factors
or an increased metabolic clearance rate. Serum levels were
significantly decreased 24 hours after stroke onset, and remained
decreased for over 10 days (Schwab, S. et al., Stroke 28:1744-1748,
1997). Serum IGF-1 may be a sensitive indicator of cerebral injury.
However, the ubiquitous expression pattern of IGF-1 indicates that
all tissues can potentially affect serum concentrations of IGF-1,
compromising the specificity of any assay using IGF-1 as a marker
for stroke. In this regard, IGF-1 may be best suited as a
cerebrospinal fluid marker of cerebral ischemia, where its dominant
source would be neural tissue.
[0208] Adhesion molecules are involved in the inflammatory response
can also be considered as acute phase reactants, as their
expression levels are altered as a result of insult. Examples of
such adhesion molecules include E-selectin, intercellular adhesion
molecule-1, vascular cell adhesion molecule, and the like.
[0209] E-selectin, also called ELAM-1 and CD62E, is a 140 kDa cell
surface C-type lectin expressed on endothelial cells in response to
IL-1 and TNF.alpha. that mediates the "rolling" interaction of
neutrophils with endothelial cells during neutrophil recruitment.
The normal serum concentration of E-selectin is approximately 50
ng/ml (2.9 nM). Investigations into the changes on serum E-selectin
concentrations following stroke have reported mixed results. Some
investigations report increases in serum E-selectin concentration
following ischemic stroke, while others find it unchanged (Bitsch,
A. et al., Stroke 29:2129-2135, 1998; Kim, J. S., J. Neurol. Sci.
137:69-78, 1996; Shyu, K. G. et al., J. Neurol. 244:90-93, 1997).
E-selectin concentrations are elevated in the CSF of individuals
with subarachnoid hemorrhage and may predict vasospasm (Polin, R.
S. et al., J. Neurosurg. 89:559-567, 1998). Elevations in the serum
concentration of E-selectin would indicate immune system
activation. Serum E-selectin concentrations are elevated in
individuals with, atherosclerosis, various forms of cancer,
preeclampsia, diabetes, cystic fibrosis, AMI, and other nonspecific
inflammatory states (Hwang, S. J. et al., Circulation 96:4219-4225,
1997; Banks, R. E. et al., Br. J. Cancer 68:122-124, 1993;
Austgulen, R. et al., Eur. J. Obstet. Gynecol. Reprod. Biol.
71:53-58, 1997; Steiner, M. et al., Thromb. Haemost. 72:979-984,
1994; De Rose, V. et al., Am. J. Respir. Crit. Care Med.
157:1234-1239, 1998). The serum concentration of E-selectin may be
elevated following ischemic stroke, but it is not clear if these
changes are transient or regulated by an as yet unidentified
mechanism. Serum E-selectin may be a specific marker of endothelial
cell injury. It is not, however, a specific marker for stroke or
cerebral injury, since it is elevated in the serum of individuals
with various conditions causing the generation of an inflammatory
state. Furthermore, elevation of serum E-selectin concentration is
associated with some of the risk factors associated with
stroke.
[0210] Head activator (HA) is an 11 amino acid, 1.1 kDa
neuropeptide that is found in the hypothalamus and intestine. It
was originally found in the freshwater coelenterate hydra, where it
acts as a head-specific growth and differentiation factor. In
humans, it is thought to be a growth regulating agent during brain
development. The normal serum HA concentration is <0.1 ng/ml
(100 pM) Serum HA concentration is persistently elevated in
individuals with tumors of neural or neuroendocrine origin
(Schaller, H. C. et al., J Neurooncol. 6:251-258, 1988; Winnikes,
M. et al., Eur. J. Cancer 28:421-424, 1992). No studies have been
reported regarding HA serum elevations associated with stroke. HA
is presumed to be continually secreted by tumors of neural or
neuroendocrine origin, and serum concentration returns to normal
following tumor removal. Serum HA concentration can exceed 6.8
ng/ml (6.8 nM) in individuals with neuroendocrine-derived tumors.
The usefulness of HA as part of a stroke panel would be to identify
individuals with tumors of neural or neuroendocrine origin. These
individuals may have serum elevations of markers associated with
cerebral injury as a result of cancer, not cerebral injury related
to stroke. Although these individuals may be a small subset of the
group of individuals that would benefit from a rapid diagnostic of
cerebral injury, the use of HA as a marker would aid in their
identification. Finally, angiotensin converting enzyme, a serum
enzyme, has the ability to degrade HA, and blood samples would have
to be drawn using EDTA as an anticoagulant to inhibit this
activity.
[0211] C-type natriuretic peptide (CNP) a 22-amino acid peptide
that is the primary active natriuretic peptide in the human brain;
CNP is also considered to be an endothelium-derived relaxant
factor, which acts in the same way as nitric oxide (NO) (Davidson
et al., Circulation 93:1155-9, 1996). CNP is structurally related
to Atrial natriuretic peptide (ANP) and B-type natriuretic peptide
(BNP); however, while ANP and BNP are synthesized predominantly in
the myocardium, CNP is synthesized in the vascular endothelium as a
precursor (pro-CNP) (Prickett et al., Biochem. Biophys. Res.
Commun. 286:513-7, 2001). CNP is thought to possess vasodilator
effects on both arteries and veins and has been reported to act
mainly on the vein by increasing the intracellular cGMP
concentration in vascular smooth muscle cells.
[0212] Adrenomedullin (AM) is a 52-amino acid peptide which is
produced in many tissues, including adrenal medulla, lung, kidney
and heart (Yoshitomi et al., Clin. Sci. (Colch) 94:135-9, 1998).
Intravenous administration of AM causes a long-lasting hypotensive
effect, accompanied with an increase in the cardiac output in
experimental animals. AM has been reported to enhance the
stretch-induced release of ANP from the right atrium, but not to
affect ventricular BNP expression. AM is synthesized as a precursor
molecule (pro-AM). The N-terminal peptide processed from the AM
precursor has also been reported to act as a hypotensive peptide
(Kuwasako et al., Ann. Clin. Biochem. 36:622-8, 1999).
[0213] The endothelins are three related peptides (endothelin-1,
endothelin-2, and endothelin-3) encoded by separate genes that are
produced by vascular endothelium, each of which exhibit potent
vasoconstricting activity. Endothelin-1 (ET-1) is a 21 amino acid
residue peptide, synthesized as a 212 residue precursor
(preproET-1), which contains a 17 residue signal sequence that is
removed to provide a peptide known as big ET-1. This molecule is
further processed by hydrolysis between trp21 and val22 by
endothelin converting enzyme. Both big ET-1 and ET-1 exhibit
biological activity; however the mature ET-1 form exhibits greater
vasoconstricting activity (Brooks and Ergul, J. Mol. Endocrinol.
21:307-15, 1998). Similarly, endothelin-2 and endothelin-3 are also
21 amino acid residues in length, and are produced by hydrolysis of
big endothelin-2 and big endothelin-3, respectively (Yap et al.,
Br. J. Pharmacol. 129:170-6, 2000; Lee et al., Blood 94:1440-50,
1999).
[0214] Urotensin 2 is a peptide having the sequence
Ala-Gly-Thr-Ala-Asp-Cys-Phe-Trp-Lys-Tyr-Cys-Val, with a disulfide
bridge between Cys6 and Cys 11. Human urotensin 2 (UTN) is
synthesized in a prepro form. Processed urotensin 2 has potent
vasoactive and cardiostimulatory effects, acting on the G
protein-linked receptor GPR14.
[0215] Vasopressin (arginine vasopressin, AVP; antidiuretic
hormone, ADH) is a peptide hormone released from the posterior
pituitary. Its primary function in the body is to regulate
extracellular fluid volume by affecting renal handling of water.
There are several mechanisms regulating release of AVP.
Hypovolemia, as occurs during hemorrhage, results in a decrease in
atrial pressure. Specialized stretch receptors within the atrial
walls and large veins (cardiopulmonary baroreceptors) entering the
atria decrease their firing rate when there is a fall in atrial
pressure. Afferent from these receptors synapse within the
hypothalamus; atrial receptor firing normally inhibits the release
of AVP by the posterior pituitary. With hypovolemia or decreased
central venous pressure, the decreased firing of atrial stretch
receptors leads to an increase in AVP release. Hypothalamic
osmoreceptors sense extracellular osmolarity and stimulate AVP
release when osmolarity rises, as occurs with dehydration. Finally,
angiotensin II receptors located in a region of the hypothalamus
regulate AVP release--an increase in angiotensin II simulates AVP
release.
[0216] Heart Failure is also associated with what might be viewed
as a paradoxical increase in AVP. Increased blood volume and atrial
pressure associated with heart failure suggest that AVP secretion
might be inhibited, but is is not. It may be that sympathetic and
renin-angiotensin system activation in heart failure override the
volume and low pressure cardiovascular receptors (as well as the
osmoregulation of AVP) and cause an increase in AVP secretion.
Nevertheless, this increase in AVP during heart failure may
contribute to the increase in systemic vascular resistance as well
as enhance renal retention of fluid.
[0217] AVP has two principle sites of action: kidney and blood
vessels. The most important physiological action of AVP is that it
increases water reabsorption by the kidneys by increasing water
permeability in the collecting duct, thereby permitting the
formation of a more concentrated urine. This is the antidiuretic
effect of AVP. This hormone also constricts arterial blood vessels;
however, the normal physiological concentrations of AVP are below
its vasoactive range.
[0218] Calcitonin gene related peptide (CGRP) is a polypeptide of
37 amino acids that is a product of the calcitonin gene derived by
alternative splicing of the precursor mRNA. The calcitonin gene
(CALC-I) primary RNA transcript is processed into different mRNA
segments by inclusion or exclusion of different exons as part of
the primary transcript. Calcitonin-encoding mRNA is the main
product of CALC-I transcription in C-cells of the thyroid, whereas
CGRP-I mRNA (CGRP=calcitonin-gene-related peptide) is produced in
nervous tissue of the central and peripheral nervous systems (FIG.
2.2.1) (9). In the third mRNA sequence, the calcitonin sequence is
lost and alternatively the sequence of CGRP is encoded in the mRNA.
CGRP is a markedly vasoactive peptide with vasodilatative
properties. CGRP has no effect on calcium and phosphate metabolism
and is synthesised predominantly in nerve cells related to smooth
muscle cells of the blood vessels (149). ProCGRP, the precursor of
CGRP, and PCT have partly identical N-terminal amino acid
sequences.
[0219] Angiotensin II is an octapeptide hormone formed by renin
action upon a circulating substrate, angiotensinogen, that
undergoes proteolytic cleavage to from the decapeptide angiotensin
I. Vascular endothelium, particularly in the lungs, has an enzyme,
angiotensin converting enzyme (ACE), that cleaves off two amino
acids to form the octapeptide, angiotensin II (AII).
[0220] All has several very important functions: Constricts
resistance vessels (via All receptors) thereby increasing systemic
vascular resistance and arterial pressure; Acts upon the adrenal
cortex to release aldosterone, which in turn acts upon the kidneys
to increase sodium and fluid retention; Stimulates the release of
vasopressin (antidiuretic hormone, ADH) from the posterior
pituitary which acts upon the kidneys to increase fluid retention;
Stimulates thirst centers within the brain; Facilitates
norepinephrine release from sympathetic nerve endings and inhibits
norepinephrine re-uptake by nerve endings, thereby enhancing
sympathetic adrenergic function; and Stimulates cardiac hypertrophy
and vascular hypertrophy.
[0221] Glycated hemoglobin HbA1c measurement provides an assessment
of the degree to which blood glucose has been elevated over an
extended time period, and so has been related to the extent
diabetes is controlled in a patient. Glucose binds slowly to
hemoglobin A, forming the A1c subtype. The reverse reaction, or
decomposition, proceeds relatively slowly, so any buildup persists
for roughly 4 weeks. With normal blood glucose levels, glycated
hemoglobin is expected to be 4.5% to 6.7%. As blood glucose
concentration rise, however, more binding occurs. Poor blood sugar
control over time is suggested when the glycated hemoglobin measure
exceeds 8.0%.
[0222] (viii) Markers Related to Apoptosis
[0223] Caspase-3, also called CPP-32, YAMA, and apopain, is an
interleukin-1 converting enzyme (ICE)-like intracellular cysteine
proteinase that is activated during cellular apoptosis. Caspase-3
is present as an inactive 32 kDa precursor that is proteolytically
activated during apoptosis induction into a heterodimer of 20 kDa
and 11 kDa subunits (Fernandes-Alnemri, T. et al., J. Biol. Chem.
269:30761-30764, 1994). Its cellular substrates include
poly(ADP-ribose) polymerase (PARP) and sterol regulatory element
binding proteins (SREBPs) (Liu, X. et al., J. Biol. Chem.
271:13371-13376, 1996). The normal plasma concentration of
caspase-3 is unknown. There are no published investigations into
changes in the plasma concentration of caspase-3 associated with
ACS. There are increasing amounts of evidence supporting the
hypothesis of apoptosis induction in cardiac myocytes associated
with ischemia and hypoxia (Saraste, A., Herz 24:189-195, 1999;
Ohtsuka, T. et al., Coron. Artery Dis. 10:221-225, 1999; James, T.
N., Coron. Artery Dis. 9:291-307, 1998; Bialik, S. et al., J. Clin.
Invest. 100:1363-1372, 1997; Long, X. et al., J. Clin. Invest.
99:2635-2643, 1997). Elevations in the plasma caspase-3
concentration may be associated with any physiological event that
involves apoptosis. There is evidence that suggests apoptosis is
induced in skeletal muscle during and following exercise and in
cerebral ischemia (Carraro, U. and Franceschi, C., Aging (Milano)
9:19-34, 1997; MacManus, J. P. et al., J. Cereb. Blood Flow Metab.
19:502-510, 1999).
[0224] Cathepsin D (E.C.3.4.23.5.) is a soluble lysosomal aspartic
proteinase. It is synthesized in the endoplasmic reticulum as a
preprocathepsin D. Having a mannose-6-phosphate tag, procathepsin D
is recognized by a mannose-6-phosphate receptor. Upon entering into
an acidic lysosome, the single-chain procathepsin D (52 KDa) is
activated to cathepsin D and subsequently to a mature two-chain
cathepsin D (31 and 14 KDa, respectively). The two
mannose-6-phosphate receptors involved in the lysosomal targeting
of procathepsin D are expressed both intracellularly and on the
outer cell membrane. The glycosylation is believed to be crucial
for normal intracellular trafficking. The fundamental role of
cathepsin D is to degrade intracellular and internalized proteins.
Cathepsin D has been suggested to take part in antigen processing
and in enzymatic generation of peptide hormones. The
tissue-specific function of cathepsin D seems to be connected to
the processing of prolactin. Rat mammary glands use this enzyme for
the formation of biologically active fragments of prolactin.
Cathepsin D is functional in a wide variety of tissues during their
remodeling or regression, and in apoptosis.
[0225] Brain .alpha. spectrin (also referred to as .alpha. fodrin)
is a cytoskeletal protein of about 284 kDa that interacts with
calmodulin in a calcium-dependent manner. Like erythroid spectrin,
brain .alpha. spectrin forms oligomers (in particular dimers and
tetramers). Brain a spectrin contains two EF-hand domains and 23
spectrin repeats. The caspase 3-mediated cleavage of a spectrin
during apoptotic cell death may play an important role in altering
membrane stability and the formation of apoptotic bodies.
[0226] Other Preferred Markers
[0227] The following table provides a list of additional preferred
markers, associated with a disease or condition for which each
marker can provide useful information for differential diagnosis.
Various markers may be listed for more than one condition. As
understood by the skilled artisan and described herein, markers may
indicate different conditions when considered with additional
markers in a panel; alternatively, markers may indicate different
conditions when considered in the entire clinical context of the
patient.
4 Marker Classification Haptoglobin Inflammatory Hepcidin Acute
phase reactant HSP-60 Acute phase reactant HSP-65 Acute phase
reactant HSP-70 Acute phase reactant Myoglobin Myocardial injury
PAPPA Inflammatory PECAM 1 Acute phase reactant
Prostaglandin-D-Synthetase Marker of ischemia S100 Myocardial
injury s-CD40 ligand* Inflammatory S-FAS ligand Acute phase
reactant Troponin I and complexes Myocardial injury cardiotrophin 1
Inflammatory urotensin II Blood pressure regulation asymmetric
dimethylarginine Acute phase reactant BNP Blood pressure regulation
Fibrinogen coagulation and hemostasis ANP Blood pressure regulation
CNP Blood pressure regulation Ubiquitin Fusion Degradation Protein
I Homolog Apoptosis alpha 2 actin Vascular tissue basic calponin 1
Vascular tissue beta like 1 integrin Vascular tissue Calponin
Vascular tissue CSRP2 Vascular tissue elastin Vascular tissue
Fibrillin 1 Vascular tissue LTBP4 Vascular tissue smooth muscle
myosin Vascular tissue transgelin Vascular tissue calcitonin gene
related peptide Blood pressure regulation Carboxyterminal
propeptide of Marker of collagen synthesis type I procollagen
(PICP) Collagen carboxyterminal Marker of collagen degradation
telopeptide (ICTP) Fibronectin Inflammatory MMP-11 Acute phase
reactant MMP-3 Acute phase reactant MMP-9 Acute phase reactant
arg-Vasopressin Blood pressure regulation aldosterone Blood
pressure regulation angiotensin 1 Blood pressure regulation
angiotensin 2 Blood pressure regulation angiotensin 3 Blood
pressure regulation Antithrombin-III coagulation and hemostasis
Bradykinin Blood pressure regulation calcitonin Blood pressure
regulation Endothelin-2 Blood pressure regulation Endothelin-3
Blood pressure regulation Renin Blood pressure regulation
Urodilatin Blood pressure regulation Defensin HBD 1 Acute phase
reactant Defensin HBD 2 Acute phase reactant alpha enolase
Pulmonary tissue specific LAMP 3 Pulmonary tissue specific LAMP3
Pulmonary tissue specific Lung Surfactant protein D Pulmonary
tissue specific phospholipase D Pulmonary tissue specific PLA2G5
Pulmonary tissue specific SFTPC Pulmonary tissue specific D-dimer
coagulation and hemostasis HMG Inflammatory IL-1 Inflammatory IL-8
Inflammatory IL-10* Inflammatory IL-11* Inflammatory IL-13*
Inflammatory IL-18* Inflammatory IL-4* Inflammatory macrophage
inhibitory factor Inflammatory s-acetyl Glutathione apoptosis Serum
Amyloid A Acute phase reactant s-iL 18 receptor pro and
anti-Inflammatory modulator S-iL-1 receptor pro and
anti-Inflammatory modulator s-TNF P55 Inflammatory and growth
factor s-TNF P75 Inflammatory and growth factor TGF-beta Acute
phase reactant MMP-11 Acute phase reactant PAI-1 coagulation and
hemostasis Procalcitonin Blood pressure regulation PROTEIN C
coagulation and hemostasis TAFI coagulation and hemostasis CRP
Acute phase reactant e-selectin Acute phase reactant 14-3-3 Neural
tissue injury 4.1B Neural tissue injury adrenomedullin Blood
pressure regulation APO E4-1 Neural tissue injury Atrophin 1 Neural
tissue injury Beta NGF Acute phase reactant beta thromboglobulin
coagulation and hemostasis BNP Blood pressure regulation brain
Derived neurotrophic Neural tissue injury factor Brain Fatty acid
binding protein Neural tissue injury brain tubulin Neural tissue
injury CACNA1A Neural tissue injury Calbindin D Neural tissue
injury Calbrain Neural tissue injury calcyphosine Blood pressure
regulation Carbonic anhydrase XI Neural tissue injury Caspase 3
apoptosis Cathepsin D apoptosis CBLN1 Neural tissue injury CD44
Inflammatory Cerebellin 1 Neural tissue injury Chimerin 1 Neural
tissue injury Chimerin 2 Neural tissue injury CHN1 Neural tissue
injury CHN2 Neural tissue injury Ciliary neurotrophic factor Neural
tissue injury CKBB Neural tissue injury CNP Blood pressure
regulation CRHR1 Neural tissue injury C-tau Neural tissue injury
cytochrome C apoptosis DRPLA Neural tissue injury EGF Inflammatory
and growth factors Endothelin-1 Blood pressure regulation
E-selectin Acute phase reactant Fibrinopeptide A coagulation and
hemostasis Fibronectin Inflammatory GFAP Neural tissue injury
Glutathione S Transferase Acute phase reactant GPM6B Neural tissue
injury GPR7 Neural tissue injury GPR8 Neural tissue injury GRIN2C
Neural tissue injury GRM7 Neural tissue injury HAPIP Neural tissue
injury HIF 1 ALPHA Acute phase reactant HIP2 Neural tissue injury
HSP-60 Acute phase reactant IL-10 Inflammatory IL-1-Beta
Inflammatory IL-1ra Inflammatory IL-6 Inflammatory IL-8
Inflammatory I-NOS Acute phase reactant Insulin-like growth factor
Inflammatory Intracellular adhesion molecule Acute phase reactant
KCNK4 Neural tissue injury KCNK9 Neural tissue injury KCNQ5 Neural
tissue injury Lactate dehydrogenase Acute phase reactant MAPK10
Neural tissue injury MCP-1 Acute phase reactant MDA-LDL plaque
rupture MMP-3 Acute phase reactant MMP-9 Acute phase reactant
myelin basic protein Neural tissue injury n-acetyl aspartate Acute
phase reactant NCAM Neural tissue injury NDPKA Neural tissue injury
Neural cell adhesion molecule Neural tissue injury NEUROD2 Neural
tissue injury Neurofiliment L Neural tissue injury Neuroglobin
Neural tissue injury neuromodulin Neural tissue injury Neuron
specific enolase Neural tissue injury Neuropeptide Y Neural tissue
injury Neurotensin Neural tissue injury Neurotrophin 1,2,3,4 Neural
tissue injury NRG2 Neural tissue injury Osteoprotegerin
Inflammatory PACE4 Neural tissue injury phosphoglycerate mutase
Neural tissue injury PKC gamma Neural tissue injury Plasmin alpha 2
antiplasmin coagulation and hemostasis complex Platelet factor 4
coagulation and hemostasis Prostaglandin D-synthase Acute phase
reactant Prostaglandin E2 Acute phase reactant proteolipid protein
Neural tissue injury PTEN Neural tissue injury PTPRZ1 Neural tissue
injury RANK ligand Acute phase reactant RGS9 Neural tissue injury
RNA Binding protein Regulatory Subunit Neural tissue injury S-100b
Neural tissue injury SCA7 Neural tissue injury secretagogin Neural
tissue injury SLC1A3 Neural tissue injury SORL1 Neural tissue
injury spectrin apoptosis SREB3 Neural tissue injury STAC Neural
tissue injury STX1A Neural tissue injury STXBP1 Neural tissue
injury Syntaxin Neural tissue injury Thrombin antithrombin III
coagulation and hemostasis complex Thrombomodulin coagulation and
hemostasis Thrombus Precursor Protein coagulation and hemostasis
Tissue factor coagulation and hemostasis TNF Receptor Superfamily
Acute phase reactant Member 1A Transforming growth factor beta
Inflammatory transthyretin Neural tissue injury Tumor necrosis
factor alpha Acute phase reactant Vascular cell adhesion molecule
Acute phase reactant Vascular endothelial growth Inflammatory
factor von Willebrand factor coagulation and hemostasis adenylate
kinase-1 Neural tissue injury BDNF* Neural tissue injury CGRP Blood
pressure regulation cystatin C Acute phase reactant neurokinin A
Neural tissue injury substance P Inflammatory D Dimer coagulation
and hemostasis Myeloperoxidase (MPO) Inflammatory Oxidized
Low-Density Lipoproteins (OxLDL) markers of atherosclerosis
[0228] Exemplary Marker Panels for Distinguishing Systolic and
Diastolic Heart Failure
[0229] Exemplary marker panels related to differentiating systolic
and diastolic function comprise one or more markers selected from
the group consisting of BNP, BNP related peptides, aldosterone,
ANP, ANP related peptides, urodilatin, angiotensin 1, angiotensin
2, angiotensin 3, bradykinin, calcitonin, calcitonin gene related
peptide, endothelin-2, endothelin-3, renin, urotensin 1, urotensin
2, antithrombin III, D-dimer, MMP-3, MMP-9, MMP-11, carboxy
terminal propeptide of type I collagen (PICP), collagen carboxy
terminal telopeptide (ICTP), fibrinogen, fibronectin, and
vasopressin. Markers related to both systolic and diastolic
dysfunction include BNP, ANP and ANP related markers. A preferred
list of markers for differentiating systolic and diastolic heart
failure include one or more markers selected from the group
consisting of BNP, BNP related peptides, calcitonin gene related
peptide, urotensin 2, endothelin 2, calcitonin and angiotensin 2. A
particularly preferred list of markers for differentiating systolic
and diastolic dysfunction include one or more markers selected from
the group consisting of BNP, angiotensin 2, urotensin 2, and
calcitonin gene related peptide.
[0230] Congestive heart failure is a heterogenous condition arising
from two primary pathologies: left ventricular diastolic
dysfunction and systolic dysfunction, which occur either alone or
in combination. Gaasch, JAMA 271: 1276-80 (1994). As many as 40
percent of patients with clinical heart failure have diastolic
dysfunction with normal systolic function. Soufer et al., Am. J.
Cardiol. 55: 1032-6 (1984). Patient care decisions and prognosis
hinge upon determination of the presence of one or both of these
pathologies. Shamsham and Mitchell, Am. Fam. Physician 2000;
61:1319-28 (2000).
[0231] Recently, BNP has been reported as a useful marker in the
diagnosis of congestive heart failure. Dao et al., J. Am. Coll.
Cardiol. 37: 379-85 (2001). However, BNP levels alone are not able
to distinguish diastolic dysfunction from systolic dysfunction.
Krishnaswamy et al., Am. J. Med. 111: 274-79 (2001).
[0232] Exemplary Marker Panels for Distinguishing Aortic
Dissection, Myocardial Ischemia and Myocardial Infarction
[0233] Exemplary marker panels related to differentiating aortic
dissection, myocardial ischemia, and myocardial infarction comprise
one or more markers selected from the group consisting of smooth
muscle myosin and/or smooth muscle myosin heavy chain (both aortic
dissection markers), BNP and/or BNP related peptides, one or more
troponin forms (myocardial ischemia and infarction), and myoglobin
(myocardial infarction or necrosis).
[0234] Exemplary Marker Panels for Distinguishing Atrial
Fibrillation, Myocardial Infarction, and/or Congestive Heart
Failure
[0235] Exemplary marker panels related to differentiating atrial
fibrillation, myocardial infarction, and/or congestive heart
failure comprise markers selected from the group consisting of ANP,
ANP related peptides (atrial fibrillation), one or more troponin
forms, myoglobin, BNP, and BNP related peptides.
[0236] Assay Measurement Strategies
[0237] Numerous methods and devices are well known to the skilled
artisan for the detection and analysis of the markers of the
instant invention. With regard to polypeptides or proteins in
patient test samples, immunoassay devices and methods are often
used. See, e.g., U.S. Pat. Nos. 6,143,576; 6,113,855; 6,019,944;
5,985,579; 5,947,124; 5,939,272; 5,922,615; 5,885,527; 5,851,776;
5,824,799; 5,679,526; 5,525,524; and 5,480,792, each of which is
hereby incorporated by reference in its entirety, including all
tables, figures and claims. These devices and methods can utilize
labeled molecules in various sandwich, competitive, or
non-competitive assay formats, to generate a signal that is related
to the presence or amount of an analyte of interest. Additionally,
certain methods and devices, such as biosensors and optical
immunoassays, may be employed to determine the presence or amount
of analytes without the need for a labeled molecule. See, e.g.,
U.S. Pat. Nos. 5,631,171; and 5,955,377, each of which is hereby
incorporated by reference in its entirety, including all tables,
figures and claims. One skilled in the art also recognizes that
robotic instrumentation including but not limited to Beckman
Access, Abbott AxSym, Roche ElecSys, Dade Behring Stratus systems
are among the immunoassay analyzers that are capable of performing
the immunoassays taught herein.
[0238] Preferably the markers are analyzed using an immunoassay,
although other methods are well known to those skilled in the art
(for example, the measurement of marker RNA levels). The presence
or amount of a marker is generally determined using antibodies
specific for each marker and detecting specific binding. Any
suitable immunoassay may be utilized, for example, enzyme-linked
immunoassays (ELISA), radioimmunoassays (RIAs), competitive binding
assays, and the like. Specific immunological binding of the
antibody to the marker can be detected directly or indirectly.
Direct labels include fluorescent or luminescent tags, metals,
dyes, radionuclides, and the like, attached to the antibody.
Indirect labels include various enzymes well known in the art, such
as alkaline phosphatase, horseradish peroxidase and the like.
[0239] The use of immobilized antibodies specific for the markers
is also contemplated by the present invention. The antibodies could
be immobilized onto a variety of solid supports, such as magnetic
or chromatographic matrix particles, the surface of an assay place
(such as microtiter wells), pieces of a solid substrate material or
membrane (such as plastic, nylon, paper), and the like. An assay
strip could be prepared by coating the antibody or a plurality of
antibodies in an array on solid support. This strip could then be
dipped into the test sample and then processed quickly through
washes and detection steps to generate a measurable signal, such as
a colored spot.
[0240] The analysis of a plurality of markers may be carried out
separately or simultaneously with one test sample. For separate or
sequential assay of markers, suitable apparatuses include clinical
laboratory analyzers such as the ElecSys (Roche), the AxSym
(Abbott), the Access (Beckman), the ADVIA.RTM. CENTAUR.RTM. (Bayer)
immunoassay systems, the NICHOLS ADVANTAGE.RTM. (Nichols Institute)
immunoassay system, etc. Preferred apparatuses or protein chips
perform simultaneous assays of a plurality of markers on a single
surface. Particularly useful physical formats comprise surfaces
having a plurality of discrete, adressable locations for the
detection of a plurality of different analytes. Such formats
include protein microarrays, or "protein chips" (see, e.g., Ng and
Ilag, J. Cell Mol. Med. 6: 329-340 (2002)) and certain capillary
devices (see, e.g., U.S. Pat. No. 6,019,944). In these embodiments,
each discrete surface location may comprise antibodies to
immobilize one or more analyte(s) (e.g., a marker) for detection at
each location. Surfaces may alternatively comprise one or more
discrete particles (e.g., microparticles or nanoparticles)
immobilized at discrete locations of a surface, where the
microparticles comprise antibodies to immobilize one analyte (e.g.,
a marker) for detection.
[0241] Several markers may be combined into one test for efficient
processing of a multiple of samples. In addition, one skilled in
the art would recognize the value of testing multiple samples (for
example, at successive time points) from the same individual. Such
testing of serial samples will allow the identification of changes
in marker levels over time. Increases or decreases in marker
levels, as well as the absence of change in marker levels, would
provide useful information about the disease status that includes,
but is not limited to identifying the approximate time from onset
of the event, the presence and amount of salvagable tissue, the
appropriateness of drug therapies, the effectiveness of various
therapies as indicated by reperfusion or resolution of symptoms,
differentiation of the various types of ACS, identification of the
severity of the event, identification of the disease severity, and
identification of the patient's outcome, including risk of future
events.
[0242] A panel consisting of the markers referenced above may be
constructed to provide relevant information related to differential
diagnosis. Such a panel may be constucted using 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 15, 20, or more or individual markers. The analysis of
a single marker or subsets of markers comprising a larger panel of
markers could be carried out by one skilled in the art to optimize
clinical sensitivity or specificity in various clinical settings.
These include, but are not limited to ambulatory, urgent care,
critical care, intensive care, monitoring unit, inpatient,
outpatient, physician office, medical clinic, and health screening
settings. Furthermore, one skilled in the art can use a single
marker or a subset of markers comprising a larger panel of markers
in combination with an adjustment of the diagnostic threshold in
each of the aforementioned settings to optimize clinical
sensitivity and specificity. The clinical sensitivity of an assay
is defined as the percentage of those with the disease that the
assay correctly predicts, and the specificity of an assay is
defined as the percentage of those without the disease that the
assay correctly predicts (Tietz Textbook of Clinical Chemistrys
2.sup.nd edition, Carl Burtis and Edward Ashwood eds., W.B.
Saunders and Company, p. 496).
[0243] The analysis of markers could be carried out in a variety of
physical formats as well. For example, the use of microtiter plates
or automation could be used to facilitate the processing of large
numbers of test samples. Alternatively, single sample formats could
be developed to facilitate immediate treatment and diagnosis in a
timely fashion, for example, in ambulatory transport or emergency
room settings.
[0244] In another embodiment, the present invention provides a kit
for the analysis of markers. Such a kit preferably comprises
devises and reagents for the analysis of at least one test sample
and instructions for performing the assay. Optionally the kits may
contain one or more means for using information obtained from
immunoassays performed for a marker panel to rule in or out certain
diagnoses.
[0245] Selecting a Treatment Regimen
[0246] Just as the potential causes of any particular nonspecific
symptom may be a large and diverse set of conditions, the
appropriate treatments for these potential causes may be equally
large and diverse. However, once a diagnosis is obtained, the
clinician can readily select a treatment regimen that is compatible
with the diagnosis. Taking just some of the causes of dyspnea for
example, initial treatment for pulmonary embolism is supportive,
involving analgesics, oxygen, and potentially .beta.-adrenergic
stimulation. Thrombolytic therapy or embolectomy may be indicated.
In contrast, treatment for systolic dysfunction in congestive heart
failure can include therapeutic amounts of ACE inhibitors, digoxin,
.alpha.-blockers, and diuretics. In particularly serious chronic
heart failure, heart transplant may be indicated. The skilled
artisan is aware of appropriate treatments for numerous diseases
discussed in relation to the methods of diagnosis described herein.
See, e.g., Merck Manual of Diagnosis and Therapy, 17.sup.th Ed.
Merck Research Laboratories, Whitehouse Station, N.J., 1999.
EXAMPLES
[0247] The following examples serve to illustrate the present
invention. These examples are in no way intended to limit the scope
of the invention.
Example 1
Blood Sampling
[0248] Blood specimens were collected by trained study personnel
using EDTA as the anticoagulant and centrifuged for greater than or
equal to 10 minutes. The plasma component was transferred into a
sterile cryovial and frozen at -20.degree. C. or colder. Specimens
from the following population of patients and normal healthy donors
were collected (Table 1). Clinical histories were available for
each of the patients to aid in the statistical analysis of the
assay data.
Example 2
Biochemical Analyses
[0249] Markers were measured using standard immunoassay techniques.
These techniques involved the use of antibodies to specifically
bind the protein targets. A monoclonal antibody directed against a
selected marker was biotinylated using N-hydroxysuccinimide biotin
(NHS-biotin) at a ratio of about 5 NHS-biotin moieties per
antibody. The antibody-biotin conjugate was then added to wells of
a standard avidin 384 well microtiter plate, and antibody conjugate
not bound to the plate was removed. This formed the "anti-marker"
in the microtiter plate. Another monoclonal antibody directed
against the same marker was conjugated to alkaline phosphatase
using succinimidyl 4-[N-maleimidomethyl]-cyclohexane-
-1-carboxylate (SMCC) and N-succinimidyl
3-[2-pyridyldithio]propionate (SPDP) (Pierce, Rockford, Ill.).
[0250] Immunoassays were performed on a TECAN Genesis RSP 200/8
Workstation. Biotinylated antibodies were pipetted into microtiter
plate wells previously coated with avidin and incubated for 60 min.
The solution containing unbound antibody was removed, and the wells
were washed with a wash buffer, consisting of 20 mM borate (pH
7.42) containing 150 mM NaCl, 0.1% sodium azide, and 0.02%
Tween-20. The plasma samples (10 .mu.L) were pipeted into the
microtiter plate wells, and incubated for 60 min. The sample was
then removed and the wells were washed with a wash buffer. The
antibody-alkaline phosphatase conjugate was then added to the wells
and incubated for an additional 60 min after which time, the
antibody conjugate was removed and the wells were washed with a
wash buffer. A substrate, (AttoPhos.RTM., Promega, Madison, Wis.)
was added to the wells, and the rate of formation of the
fluorescent product was related to the concentration of the marker
in the patient samples.
Example 3
Dyspnea Analysis
[0251] The following table compares levels of pulmonary surfactant
protein D, D-dimer, BNP, total cardiac troponin I, and the ratio of
BNP:D-dimer in individual patients presenting with clinical dyspnea
and in normal subjects. Dyspnea patients were subdivided into
patients receiving a clinical diagnosis of congestive heart
failure, and those receiving a clinical diagnosis of pulmonary
embolism. All units are ng/ml except BNP (pg/ml) and ratios.
5 Multi-Center CHF Patients Patient ID PSD D-Dimer BNP Tnl Ratio 16
35.4 88 889 2.1 10.1 012 8.7 113 1228 2.2 10.9 Moore 003 6.9 62 552
0.0 8.9 11 11.7 160 987 0.5 6.2 010 13.3 145 466 0.0 3.2 18 7.9 39
330 0.0 8.6 131-2 7.2 125 1031 0.0 8.3 125-1 3.7 49 314 0.0 6.4 115
8.1 203 185 0.0 0.9 128-1 7.5 141 228 0.0 1.6 143-1 5.1 169 402 0.0
2.4 134-1 1.9 142 251 0.0 1.8 138-1 2.4 40 521 0.0 13.0 157-1 4.1
107 231 0.0 2.2 176-1 2.6 70 234 0.0 3.4 175-1 4.6 154 498 0.0 3.2
22 6.7 36 650 0.0 18.3 21 3.9 149 453 0.0 3.0 23 11.5 147 1024 0.0
7.0 103-2 3.3 70 640 0.0 9.2 20 2.9 78 858 0.0 11.0 148-2 6.2 79
1614 0.0 20.4 173-2 2.7 68 236 0.0 3.5 166-1 5.5 53 681 0.0 12.9
178-1 4.3 89 250 0.0 2.8 183-2 9.3 109 1199 0.0 11.0 189-2 2.2 270
335 0.0 1.2 42 3.5 143 846 0.0 5.9 43 4.2 63 287 0.0 4.5 54 3.5 51
302 0.0 5.9 25 4.6 61 768 0.0 12.5 53 4.5 77 1813 0.0 23.5 59 20.8
77 288 0.0 3.7 55 2.4 53 237 0.0 4.5 158-1 2.3 53 1030 0.0 19.6
Mean 6.7 100.9 624.5 0.1 7.8 Median 4.6 79.3 498.0 0.0 6.2 St. Dev.
6.3 53.1 415.8 0.5 5.9
[0252]
6 Patient ID PSD D-Dimer BNP Tnl Ratio Multi-Center Patients with
PE 81 4.9 145 314.7 0.0 2.2 110 4.3 87 24.3 0.0 0.3 112 6.9 105
15.9 0.0 0.2 119 10.1 104 175.7 0.0 1.7 142 7.0 106 6.2 0.0 0.1 196
8.2 127 5.0 0.1 0.0 801-2 5.2 113 19.7 0.0 0.2 377-2 1.4 97 57.2
0.0 0.6 008264 1.3 258 121.3 0.0 0.5 008557 17.5 126 51.3 0.0 0.4
010647 3.8 106 355.3 0.0 3.4 10640 0.7 43 9.2 0.0 0.2 7329 3.4 191
287.3 0.0 1.5 008605 6.0 82 733.5 0.0 9.0 Mean 5.8 120.7 155.5 0.0
1.4 Median 5.0 105.7 54.3 0.0 0.4 St. Dev. 4.3 51.7 207.6 0.0 2.4
Normal Subjects 001511 5.1 90 20.4 0.0 0.2 001515 1.9 36 8.9 0.0
0.2 001520 1.0 61 6.5 0.0 0.1 001521 4.6 72 3.8 0.0 0.1 001524 2.3
69 11.1 0.0 0.2 001607 3.6 72 23.4 0.0 0.3 001610 1.1 52 18.3 0.0
0.4 001613 0.2 40 0.0 0.0 0.0 001616 2.6 28 0.0 0.0 0.0 001619 0.3
44 0.0 0.0 0.0 001622 1.4 25 0.0 0.0 0.0 001625 4.6 142 0.0 0.0 0.0
001628 1.6 40 0.0 0.0 0.0 001631 4.6 57 0.0 0.0 0.0 001634 7.2 60
6.6 0.0 0.1 001637 5.5 55 0.0 0.0 0.0 001640 0.0 260 19.1 0.0 0.1
001643 2.5 50 7.7 0.0 0.2 001646 0.0 56 4.7 0.0 0.1 002202 1.0 59
27.4 0.0 0.5 002205 1.7 39 23.4 0.0 0.6 002208 1.1 25 25.9 0.0 1.0
002211 0.9 55 45.9 0.0 0.8 002214 0.0 97 23.4 0.0 0.2 002217 2.8
117 15.3 0.0 0.1 002220 0.3 55 11.3 0.0 0.2 002223 2.5 47 8.1 0.0
0.2 002228 2.2 44 24.3 0.0 0.5 002229 2.6 61 11.2 0.0 0.2 002232
0.7 69 10.5 0.0 0.2 002235 0.0 54 4.0 0.0 0.1 002238 1.5 53 9.6 0.0
0.2 002241 7.5 16 10.8 0.0 0.7 002244 8.6 44 10.7 0.0 0.2 002247
3.7 68 33.1 0.0 0.5 Mean 2.5 63.3 12.2 0.0 0.2 Median 1.9 55.0 10.5
0.0 0.2 St. Dev. 2.3 42.3 11.1 0.0 0.3
[0253] These data indicate that the median D-dimer levels in the
patients diagnosed with pulmonary embolism is higher than for the
CHF patients, which is itself higher than normal subjects.
Pulmonary surfactant protein D levels appears to be elevated over
normals to nearly the same extent in both disease groups compared
to normals. Using <82 .mu.g/ml d-dimer as the rule-out cutoff
for a diagnosis of pulmonary embolism would result in one false
negative diagnosis, and would correctly rule out 18 of the 35 CHF
patients and 30 of the 35 normals. For this patient population,
using a d-dimer/BNP ratio of >3.4 as the rule-out cutoff would
again result in one false negative diagnosis, but would correctly
rule out 25 of the 35 CHF patients. The low cardiac troponin I
level in all disease and normal subjects correctly rules out the
occurrence of myocardial infarction in the entire test population.
This example demonstrates that the differential diagnosis of causes
of dyspnea can be accomplished through the measurement of d-dimer,
BNP and cardiac troponin. Additionally, pulmonary embolism can be
ruled in when BNP, d-dimer and pulmonary surfactant protein D
levels are elevated above normal levels and troponin levels are
normal. Pulmonary embolism can be ruled out when d dimer levels are
in the normal range. When BNP levels are above normal, one can rule
in congestive heart failure. When cardiac troponin levels are above
normal, cardiac ischemia and necrosis can be ruled in.
Example 4
Identification of Diastolic Dysfunction
[0254] The following table compares levels of BNP, vasopressin,
endothelin-2, calcitonin gene related peptide, urotensin 2, ANP,
angiotensis 2, the ratios of BNP:CGRP, BNP:ANP, BNP:urotensin 2,
and calcitonin in heart disease patients and normal subjects. The
heart disease patients are subdivided according to the New York
Heart Association classification of functional capacity and
objective assessment. See, Nomenclature and Criteria for Diagnosis
of Diseases of the Heart and Great Vessels. 9th ed. Boston, Mass:
Little, Brown & Co; 1994, pp. 253-256. The classification is
made as follows:
7 Class Functional Capacity Objective Assessment NYHA1 Patients
with cardiac disease but No objective evidence without resulting
limitation of physical of cardiovascular activity. Ordinary
physical activity does disease. not cause undue fatigue,
palpitation, dyspnea, or anginal pain. NYHA2 Patients with cardiac
disease resulting in Objective evidence slight limitation of
physical activity. of minimal They are comfortable at rest.
Ordinary cardiovascular disease. physical activity results in
fatigue, palpitation, dyspnea, or anginal pain. NYHA3 Patients with
cardiac disease resulting Objective evidence of in marked
limitation of physical moderately severe activity. They are
comfortable at rest. cardiovascular disease. Less than ordinary
activity causes fatigue, palpitation, dyspnea, or anginal pain.
NYHA4 Patients with cardiac disease resulting Objective evidence of
in inability to carry on any physical severe cardiovascular
activity without discomfort. Symptoms disease. of heart failure or
the anginal syndrome may be present even at rest. If any physical
activity is undertaken, discomfort is increased.
[0255] DD indicates patients having a clinical diagnosis of
diastolic dysfunction, and exhibit an ejection fraction of >50%.
Low ejection fraction (EF) patients are those exhibiting an
ejection fraction of <50%, and are NYHA4 class patients
considered to exhibit systolic, rather than diastolic, dysfunction.
All units are ng/ml except BNP (pg/ml) and ratios, and N is the
number of subjects in each group.
8 TABLE 4 BNP/ BNP/ BNP/ Angiotensin BNP Vasopressin Endothelin 2
CGRP CGRP Calcitonin Urotensin 2 U2 ANP ANP 2 N Normal 0 0.98 3.60
0.94 0 0.14 14.6 0 1.84 0 0.09 20 DD NYHA1 142 1.13 4.63 1.06 247
0.20 18.9 11 2.11 162 0.09 2 DD NYHA2 152 0.89 3.70 0.77 289 0.19
18.4 9 1.48 113 0.08 6 DD NYHA3 325 0.84 3.72 1.02 309 0.16 18.8 16
1.95 149 0.09 6 DD NYHA4 600 0.99 4.38 0.66 853 0.17 19.8 30 1.07
297 0.06 6 DD All 262 0.89 3.91 0.77 366 0.19 19.0 14 1.50 177 0.08
Low EF 839 1.10 4.38 0.97 957 0.19 28.0 41 2.17 413 0.07 20
[0256] These data indicate that Urotensin-2 and ANP can distinguish
diastolic dysfunction from systolic dysfunction. In both cases, the
levels are higher in systolic dysfunction than in diastolic
dysfunction. Moreover, with the addition of BNP, the the ability to
discriminate diastolic dysfunction from systolic dysfunction is
enhanced, as elevation of both BNP and ANP appears to be indicative
of systolic dysfunction while elevation of BNP with ANP at or below
normal levels appears to be indicative of diastolic dysfunction.
Urotensin 2 shows a similar pattern. CGRP contributes to the
ability to distinguish diastolic from systolic dysfunction when
expressed as a ratio with BNP where the ratio is greater in cases
of systolic dysfunction relative to diastolic dysfunction.
Example 5
Marker Panels for Caridac Differential Diagnosis
[0257] Exemplary marker panels were selected initially comprising a
marker related to blood pressure regulation and a plurality of
markers related to myocardial injury in order to develop a panel
for diagnosing and/or distinguishing congestive heart failure,
acute coronary syndromes, and myocardial infarction, and for
guiding therapy in response to the results of the assay. For this
purpose, BNP, cardiac troponin I (free and complexed), creatine
kinase-MB, and myoglobin were selected. Threshold levels for
comparison of measured marker concentrations were established in
this example using the upper end of normal values for CKMB (4.3
ng/mL), myoglobin (107 ng/mL) and troponin 1 (0.4 ng/mL). Elevation
and/or Temporal changes in these three markers, coupled with chest
pain for a period of at least 20 minutes is highly indicative of
myocardial infarction. In addition, BNP concentrations in excess of
80 pg/mL BNP can provide additional risk stratification in these
patients, as this level of BNP is related to increased rates of
death, myocardial infarction, and congestive heart failure in
comprarison to patients having a BNP level below this threshold.
Moreover, even in subjects experiencing no clinical symptoms of
disease, a BNP level in excess of 100 pg/mL is associated with a
substantially higher incidence of congestive heart failure. Thus,
this multimarker strategy can provide substantially more clinically
relevant information than can individual markers.
[0258] The addition of other markers to the multimarker panel can
provide additional clinical information for both risk
stratification and differential diagnosis. For example, D-dimer may
be added to the panel as a marker of coagulation and hemostasis. As
discussed above, the addition of D-dimer can permit the
differentiation of pulmonary embolism and/or deep venous thrombosis
from myocardial infarction and congestive heart failure, despite
the fact that the subjects may present to the clinician with
substantially similar symptoms. In this case, a threshold level of
about about 1 .mu.g/mL may be established. In addition, or in the
alternative, to D-dimer, C-reactive protein, a relatively
nonspecific indicator of inflammation, can provide additional risk
stratification to the panel.
[0259] As the number of markers in a panel increases, the
determination of a single panel response and its correlation to
various disease states by the methods described herein can be
advantageous. An example of such a panel may include rdiac troponin
I (free and complexed), creatine kinase-MB, and myoglobin, where
none of the markers are compared to a predetermined threshold.
Instead, of values for each marker within a window are assigned a
relative index (e.g., a value between 0 and 1). This window can be
defined by a midpoint and a window width (e.g., for troponin I, a
midpoint of 1.16 ng/mL with a window extending from 0.25 ng/mL to
2.06 ng/mL can be used; troponin I concentrations less than 0.25
are assigned an index value of 0, concentrations greater than 2.06
are assigned an index value of 1, and concentrations in the window
are assigned a relative value between 0 and 1, using a linear
interpolation across the window. This is repeated for each of CK-MB
and myoglobin, with different midpoints and window widths
determined according to the methods described herein. Finally, each
marker may be assigned a weighting factor, by which the index value
is multiplied. The three index values are added, and used to
determine a panel response, which may be correlated to diagnosis
(e.g., of acute myocardial infarction). Additional markers may be
included, which may include single concentrations of markers, or
may include relative changes in markers over time (e.g., an
increase in troponin I, CK-MB and/or myoglobin concentration over
60-240 minutes may act as a marker in the panel).
[0260] In the case of acute myocardial infarction, panels, window
values, and weighting factors are selected that, using a panel
response value, preferably provide a sensitivity of at least 80% at
greater than 90% specificity.
Example 5
Marker Panels for Cerebrovascular Differential Diagnosis
[0261] In the case of cerebrovascular differential diagnosis,
marker panels were selected comprising a marker related to blood
pressure regulation and a plurality of markers related to neural
tissue injury in order to develop a panel for diagnosing and/or
distinguishing stroke from patients referred to herein as "stroke
mimics." Additional classes of markers tested to increase marker
panel response include markers of apoptosis, markers of
inflammation, and/or acute phase reactants. A final exemplary panel
was identified that provided a sensitivity of at least 80% at
greater than 90% specificity. The details of this panel are
described in the following table. For each marker, window values
are defined as a window midpoint ("Midpoint"), and a window width
("Window"), which when multiplied by the midpoint provide the
+/-range for the window width. A positive window width and
weighting indicate a positive correlation of the marker with a
diagnosis of stroke (that is, the marker level is increased in the
stroke group relative to the mimic group); while a negative window
width and weighting indicate a negative correlation of the marker
with a diagnosis of stroke. Again, none of the markers are compared
to a predetermined threshold. Instead, a panel response value is
obtained as described herein.
9 Midpoint Window Weight NCAM 81.610 ng/mL -0.535 -0.203 Caspase-3
10.528 ng/mL 0.422 0.172 IL-8 54.727 pg/mL 0.637 0.078 CK-BB 3.232
ng/mL 0.559 0.133 CRP 90.283 .mu.g/mL 0.613 0.163 S-100b 214.559
pg/mL 0.409 0.112 BNP 302.561 pg/mL 0.465 0.092 MMP-9 473.153 ng/mL
0.590 0.046
[0262]
10 Analyte panel size 8 Stroke Type All stroke Ischemic stroke
Hemorrhagic stroke Total Controls 49 49 49 Disease group 48 41 7
ROC Area 0.956 0.969 0.878 Sens @ 92.5% Spec 93.8% 95.1% 85.7% Spec
@ 92.5% Sens 95.9% 95.9% 22.4% Runs (N) 100 100 100 Ave ROC Area
0.927 0.935 0.881 SD 0.018 0.024 0.039 Ave Sens @ 92.5% Spec 85.6%
86.8% 78.7% SD 6.50% 7.20% 9.60% Ave Spec @ 92.5% Sens 83.2% 84.4%
39.8% SD 8.10% 8.90% 27.80%
[0263] In addition, panels were assessed for the ability to
identify severity of neurologic deficit in stroke patients. In
these panels, controls were subjects exhibiting an NIH stroke scale
("NIHSS") score of <5, while the disease subjects exhibited an
NIHSS score of 5. As shown in the following table, simply changing
the window parameters and weighting, while using the same eight
markers described above, can provide important information about
the severity of neurologic deficit.
11 Panel 1 Panel 2 Win- Win- Midpoint dow Weight Midpoint dow
Weight CRP CRP 15.229 .mu.g/mL 0.577 0.159 21.973 .mu.g/mL 0.573
0.180 BNP BNP 254.365 pg/mL 0.408 0.121 240.978 pg/mL 0.438 0.118
CK-BB IL-8 0.370 ng/mL 0.280 0.074 41.354 pg/mL 0.519 0.149 IL-8
MMP-9 36.565 pg/mL 0.382 0.136 438.778 ng/mL -0.253 -0.117
Caspase-3 CK-BB 6.552 ng/mL 0.482 0.103 0.847 ng/mL 0.347 0.060
MMP-9 Caspase-3 445.855 ng/mL -0.327 -0.122 6.497 ng/mL 0.574 0.095
S-100b S-100b 5.248 pg/mL 0.009 -0.023 144.698 pg/mL 0.244 0.042
NCAM NCAM 66.384 ng/mL -0.264 -0.100 58.061 ng/mL -0.202 -0.069
[0264]
12 Panel 1 Panel 2 Total Controls 66 66 Disease group 24 24 ROC
Area 0.966 0.963 Sens @ 92.5% Spec 100.0% 100.0% Spec @ 92.5% Sens
95.5% 95.5% Runs (N) 100 100 Ave ROC Area 0.928 0.929 SD 0.021
0.018 Ave Sens @ 92.5% 79.6% 80.2% Spec SD 12.70% 13.30% Ave Spec @
92.5% 86.9% 87.7% Sens SD 6.80% 6.10%
[0265] Interestingly, MMP-9 shows a negative correlation with
neurologic deficit, indicating that, while MMP-9 is increased in
stroke patients relative to mimics, MMP-9 is actually decreased in
the case of stroke patients exhibiting an increased neurologic
deficit, relative to subjects with less severe neurologic deficit.
High MMP-9 may be indicative of increased revascularization, and
therefore may be a marker of positive prognosis in stroke patients.
In addition, thrombolytic treatment may be less advantageous in
stroke patients with high MMP-9, as revascularization is providing
additional perfusion of the lesion. Such panels may provide
prognostic information in diseases and procedures that are
associated with a risk of neurologic deficit. Such procedures
include carotid endarterectomy, hypothermic circulatory arrest,
aortic valve replacement, mitral valve replacement, coronary artery
surgery, endograft repair of aortic aneurism, coronary artery
bypass graft surgery, laryngeal mask insertion, and repair of
congenital heart defects.
[0266] Additional panels may be provided that utilize fewer
markers, with moderate loss of sensitivity and specificity, as
shown in the following tables:
13 Panel 3 Panel 4 Panel 5 Panel 6 Midpoint Window Weight Midpoint
Window Weight Midpoint Window Weight Midpoint Window Weight CRP CRP
CRP CRP 20.232 0.572 0.195 31.980 0.517 0.213 26.976 0.580 0.258
28.064 0.515 0.292 BNP BNP BNP BNP 257.043 0.390 0.143 235.623
0.284 0.160 214.202 0.294 0.199 262.398 0.277 0.260 MMP-9 CK-BB
CK-BB Caspase-3 -545.945 -0.335 -0.169 0.567 0.474 0.086 0.832
0.483 0.126 7.753 0.592 0.186 CK-BB Caspase-3 Caspase-3 CK-BB 0.990
0.441 0.083 6.825 0.522 0.120 7.699 0.654 0.177 0.166 0.373 0.100
Caspase-3 MMP-9 MMP-9 7.508 0.595 0.106 -464.054 -0.362 -0.156
-518.648 -0.443 -0.188 IL-8 IL-8 37.149 0.537 0.145 43.924 0.463
0.174 S-100b 182.184 0.442 0.079 81 86 86 86 26 27 27 27 0.941
0.947 0.922 0.900 100.0% 96.3% 85.2% 66.7% 92.6% 93.0% 87.2% 84.9%
100 100 100 100 0.913 0.910 0.895 0.878 0.021 0.019 0.014 0.016
74.8% 73.4% 65.3% 61.0% 12.60% 10.50% 7.90% 6.00% 83.6% 83.7% 80.9%
76.1% 6.80% 5.50% 5.10% 7.40%
[0267] While the invention has been described and exemplified in
sufficient detail for those skilled in this art to make and use it,
various alternatives, modifications, and improvements should be
apparent without departing from the spirit and scope of the
invention.
[0268] One skilled in the art readily appreciates that the present
invention is well adapted to carry out the objects and obtain the
ends and advantages mentioned, as well as those inherent therein.
The examples provided herein are representative of preferred
embodiments, are exemplary, and are not intended as limitations on
the scope of the invention. Modifications therein and other uses
will occur to those skilled in the art. These modifications are
encompassed within the spirit of the invention and are defined by
the scope of the claims.
[0269] It will be readily apparent to a person skilled in the art
that varying substitutions and modifications may be made to the
invention disclosed herein without departing from the scope and
spirit of the invention.
[0270] All patents and publications mentioned in the specification
are indicative of the levels of those of ordinary skill in the art
to which the invention pertains. All patents and publications are
herein incorporated by reference to the same extent as if each
individual publication was specifically and individually indicated
to be incorporated by reference.
[0271] The invention illustratively described herein suitably may
be practiced in the absence of any element or elements, limitation
or limitations which is not specifically disclosed herein. Thus,
for example, in each instance herein any of the terms "comprising",
"consisting essentially of" and "consisting of" may be replaced
with either of the other two terms. The terms and expressions which
have been employed are used as terms of description and not of
limitation, and there is no intention that in the use of such terms
and expressions of excluding any equivalents of the features shown
and described or portions thereof, but it is recognized that
various modifications are possible within the scope of the
invention claimed. Thus, it should be understood that although the
present invention has been specifically disclosed by preferred
embodiments and optional features, modification and variation of
the concepts herein disclosed may be resorted to by those skilled
in the art, and that such modifications and variations are
considered to be within the scope of this invention as defined by
the appended claims.
[0272] Other embodiments are set forth within the following claims.
Sequence CWU 1
1
3 1 108 PRT Homo sapiens 1 His Pro Leu Gly Ser Pro Gly Ser Ala Ser
Asp Leu Glu Thr Ser Gly 1 5 10 15 Leu Gln Glu Gln Arg Asn His Leu
Gln Gly Lys Leu Ser Glu Leu Gln 20 25 30 Val Glu Gln Thr Ser Leu
Glu Pro Leu Gln Glu Ser Pro Arg Pro Thr 35 40 45 Gly Val Trp Lys
Ser Arg Glu Val Ala Thr Glu Gly Ile Arg Gly His 50 55 60 Arg Lys
Met Val Leu Tyr Thr Leu Arg Ala Pro Arg Ser Pro Lys Met 65 70 75 80
Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Asp Arg Ile Ser Ser 85
90 95 Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His 100 105 2 134
PRT Homo sapiens 2 Met Asp Pro Gln Thr Ala Pro Ser Arg Ala Leu Leu
Leu Leu Leu Phe 1 5 10 15 Leu His Leu Ala Phe Leu Gly Gly Arg Ser
His Pro Leu Gly Ser Pro 20 25 30 Gly Ser Ala Ser Asp Leu Glu Thr
Ser Gly Leu Gln Glu Gln Arg Asn 35 40 45 His Leu Gln Gly Lys Leu
Ser Glu Leu Gln Val Glu Gln Thr Ser Leu 50 55 60 Glu Pro Leu Gln
Glu Ser Pro Arg Pro Thr Gly Val Trp Lys Ser Arg 65 70 75 80 Glu Val
Ala Thr Glu Gly Ile Arg Gly His Arg Lys Met Val Leu Tyr 85 90 95
Thr Leu Arg Ala Pro Arg Ser Pro Lys Met Val Gln Gly Ser Gly Cys 100
105 110 Phe Gly Arg Lys Met Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly
Cys 115 120 125 Lys Val Leu Arg Arg His 130 3 12 PRT Homo sapiens 3
Ala Gly Thr Ala Asp Cys Phe Trp Lys Tyr Cys Val 1 5 10
* * * * *